Methods for detecting cystic fibrosis mutations using mitra tip extraction

ABSTRACT

The present disclosure provides methods for determining whether a patient exhibiting cystic fibrosis symptoms, or a patient at risk for cystic fibrosis, will benefit from treatment with one or more anti-cystic fibrosis therapeutic agents. These methods are based on detecting hereditary cystic fibrosis related mutations in small-volume dried biological fluid samples that are collected using a volumetric absorptive microsampling device. Kits for use in practicing the methods are also provided.

TECHNICAL FIELD

The present disclosure provides methods for determining whether apatient exhibiting cystic fibrosis symptoms, or a patient at risk forcystic fibrosis, will benefit from treatment with one or moreanti-cystic fibrosis therapeutic agents. These methods are based ondetecting hereditary cystic fibrosis related mutations in small-volumedried biological fluid samples that are collected using a volumetricabsorptive microsampling device. Alterations in target nucleic acidsequences corresponding to one or more cystic fibrosis related mutationsmay be detected using next generation sequencing (NGS). Kits for use inpracticing the methods are also provided.

BACKGROUND

The following description of the background of the present disclosure isprovided simply to aid the reader in understanding the disclosure and isnot admitted to describe or constitute prior art to the presentdisclosure.

Cystic fibrosis (CF) is the most common severe autosomal recessivegenetic disorder in the Caucasian population. It affects approximately 1in 2,500 live births in North America (Boat et al., The Metabolic Basisof Inherited Disease, 6th ed., pp 2649-2680, McGraw Hill, NY (1989)).Approximately 1 in 25 persons of northern European Caucasian descent arecarriers of the disease. The responsible gene has been localized to a250,000 base pair genomic sequence present on the long arm of chromosome7. This sequence encodes a membrane-associated protein called the“cystic fibrosis transmembrane regulator” (or “CFTR”). There are greaterthan 1000 different mutations in the CFTR gene, each having varyingfrequencies of occurrence in different populations, presently reportedto the Cystic Fibrosis Genetic Analysis Consortium. These mutationsexist in both the coding regions (e.g., ΔF508, a mutation found in about70% of CF alleles, represents a deletion of a phenylalanine residue atposition 508) and the non-coding regions (e.g., the 5T, 7T, and 9Tvariants correspond to a sequence of 5, 7, or 9 thymidine bases locatedat the splice branch/acceptor site of intron 8) of the CFTR gene.

The major symptoms of cystic fibrosis include chronic pulmonary disease,pancreatic exocrine insufficiency, and elevated sweat electrolytelevels. The symptoms are consistent with cystic fibrosis being anexocrine disorder. Although recent advances have been made in theanalysis of ion transport across the apical membrane of the epitheliumof CF patient cells, it is not clear that the abnormal regulation ofchloride channels represents the primary defect in the disease.

Next-generation sequencing (NGS) is extensively used in diagnostics ofgenetic disorders, including cystic fibrosis, due to its high datathroughput and ability to detect multiple gene alterations in a singleassay. However, the procedures associated with collecting and preparingnucleic acids from biological samples (e.g., blood) are usuallycumbersome, and often require specialized equipment or technical skill.Further, certain patient groups, such as the elderly or infants, areunable to provide large volumes of blood for recurrent testing.

Thus, there is a need for rapid and non-invasive methods for determiningwhether a patient has a genetic basis for developing cystic fibrosis oris at risk of producing offspring that will suffer from cystic fibrosis.

SUMMARY

In one aspect, the present disclosure provides a method for detecting atleast one mutation in a sample CFTR nucleic acid comprising (a)extracting the sample CFTR nucleic acid from a dried biological fluidsample eluted from an absorbent tip of a microsampling device; (b)generating a library comprising amplicons corresponding to a pluralityof target segments of the sample CFTR nucleic acid; and (c) detecting atleast one mutation in at least one of the amplicons in the library usinghigh throughput massive parallel sequencing.

Additionally or alternatively, in some embodiments, the dried biologicalfluid sample is dried plasma, dried serum, or dried whole blood. In someembodiments, the dried biological fluid sample on the absorbent tip ofthe microsampling device is collected from a patient via fingerstick. Insome embodiments, the microsampling device is a volumetric absorbentmicrosampling device. In certain embodiments, the microsampling deviceis a MITRA® tip.

Additionally or alternatively, in some embodiments, elution of the driedbiological fluid sample is performed by contacting the absorbent tip ofthe microsampling device with a lysis buffer and Proteinase K. Incertain embodiments, the lysis buffer comprises guanidine hydrochloride,Tris.Cl, EDTA, Tween 20, and Triton X-100. In a further embodiment, thelysis buffer comprises 800 mM guanidine hydrochloride; 30 mM Tris.Cl, pH8.0; 30 mM EDTA, pH 8.0; 5% Tween 20; and 0.5% Triton X-100. In otherembodiments, the lysis buffer comprises 2.5-10% sodium dodecyl sulphate.

Additionally or alternatively, in some embodiments, elution of the driedbiological fluid sample is performed by contacting the absorbent tip ofthe microsampling device with the lysis buffer for up to 15 minutes at90° C. In certain embodiments, elution of the dried biological fluidsample is performed by contacting the absorbent tip of the microsamplingdevice with Proteinase K for up to 1 hour at 56° C. In otherembodiments, elution of the dried biological fluid sample is performedby contacting the absorbent tip of the microsampling device withProteinase K for up to 16-18 hours at 56° C.

In some embodiments, the sample volume of the microsampling device is nomore than 10-20 μL. In some embodiments, no more than 400 ng of genomicDNA is eluted from the absorbent tip of the microsampling device. Inother embodiments, about 100 ng to about 400 ng of genomic DNA is elutedfrom the absorbent tip of the microsampling device. In some embodiments,the method further comprises ligating an adapter sequence to the ends ofthe plurality of amplicons. The adapter sequence may be a P5 adapter, P7adapter, P1 adapter, A adapter, or Ion Xpress™ barcode adapter.Additionally or alternatively, in some embodiments, the method furthercomprises hybridizing one or more bait sequences to one or more targetsegments of the sample CFTR nucleic acid.

Additionally or alternatively, in some embodiments, the at least onemutation is selected from among a base change, a gene deletion and agene duplication. Additionally or alternatively, in some embodiments,the at least one mutation is associated with cystic fibrosis, and mayinclude one or more mutations listed in Table 2.

In any of the above embodiments, the dried biological fluid sample isobtained from an individual exhibiting cystic fibrosis symptoms, orhaving a family history of cystic fibrosis or a CFTR mutation. In someembodiments, the dried biological fluid sample is obtained from a malepartner of an obstetrics and gynecology patient having cystic fibrosisor at least one CFTR mutation.

Additionally or alternatively, in some embodiments, the plurality oftarget segments, taken together, span all coding and non-coding regionsof the CFTR gene. In some embodiments, the plurality of target segmentsfurther span about 1000 nucleotides of a promoter region immediatelyupstream of the first exon of the CFTR gene. In some embodiments, theplurality of target segments further span about 200 to 350 nucleotidesimmediately downstream of the CFTR gene.

Additionally or alternatively, in some embodiments, the high throughputmassive parallel sequencing is performed using pyrosequencing,reversible dye-terminator sequencing, SOLiD sequencing, Ionsemiconductor sequencing, Helioscope single molecule sequencing,sequencing by synthesis, sequencing by ligation, or SMRT™ sequencing. Incertain embodiments, the high throughput massive parallel sequencinginvolves a read depth approach. Additionally or alternatively, in someembodiments, the plurality of amplicons further comprise a unique indexsequence.

In another aspect, the present disclosure provides a method fordetecting at least one mutation in a sample CFTR nucleic acid comprisinggenerating a library comprising amplicons corresponding to a pluralityof target segments of the sample CFTR nucleic acid, wherein the sampleCFTR nucleic acid is extracted from a dried biological fluid sampleeluted from an absorbent tip of a microsampling device with a lysisbuffer and Proteinase K. In a further embodiment, the at least onemutation in the sample CFTR nucleic acid is detected using highthroughput massive parallel sequencing. In some embodiments, the lysisbuffer comprises guanidine hydrochloride, Tris.Cl, EDTA, Tween 20, andTriton X-100.

Additionally or alternatively, in some embodiments, the plurality oftarget segments of the sample CFTR nucleic acid comprise at least onealteration compared to the corresponding region of a reference CFTRnucleotide sequence. In certain embodiments, the reference CFTRnucleotide sequence comprises a wild-type CFTR nucleic acid sequence.

In one aspect, the present disclosure provides a method for selecting apatient exhibiting cystic fibrosis symptoms, or a patient at risk forcystic fibrosis for treatment with an anti-cystic fibrosis therapeuticagent comprising (a) eluting a dried biological fluid sample of thepatient from an absorbent tip of a microsampling device, wherein thedried biological fluid sample comprises a sample CFTR nucleic acid; (b)generating a library comprising amplicons corresponding to a pluralityof target segments of the sample CFTR nucleic acid; (c) detecting atleast one mutation in at least one of the amplicons in the library usinghigh throughput massive parallel sequencing; and (d) selecting thepatient for treatment with an anti-cystic fibrosis therapeutic agent.The dried biological fluid sample may be dried plasma, dried serum, ordried whole blood. In some embodiments, the microsampling device is avolumetric absorbent microsampling device. In certain embodiments, thedried biological fluid sample on the absorbent tip of the microsamplingdevice is collected from a patient via fingerstick. In certainembodiments, the microsampling device is a MITRA® tip. In someembodiments, the patient harbors one or more mutations in the CFTR geneand may include one or more mutations listed in Table 2.

In any of the above embodiments, the anti-cystic fibrosis therapeuticagent is one or more agents selected from the group consisting ofpenicillin, amoxicillin, cephalosporins, macrolides, fluoroquinolones,sulfonamides, Tetracyclines, aminoglycosides, colistin, Amcinonide,Betamethosone diproprionate, Clobetasol, Clocortolone, Dexamethasone,Diflorasone, Dutasteride, Flumethasone Pivalate, Flunisolide,Fluocinolone Acetonide, Fluocinonide, Fluorometholone, Fluticasonepropionate, Fluticasone propionate, Fluticasone propionate,Flurandrenolide, Hydroflumethiazide, aceclofenac, acemetacin, aspirin,celecoxib, dexibuprofen, dexketoprofen, diclofenac, etodolac,etoricoxib, fenoprofen, flurbiprofen, ibuprofen, indometacin,ketoprofen, mefenamic acid, meloxicam, nabumetone, naproxen, sulindac,tenoxicam, tiaprofenic acid, expectorants, antihistamines, coughsuppressants, Dextromethorphan, hypertonic salines, dornase alfa,mucolytics, pancreatic enzymes, vitamin A, vitamin D, vitamin E, vitaminK, and supplements reduce stomach acid.

In another aspect, the present disclosure provides a method fordetecting a genetic basis for being affected with cystic fibrosis, orfor being a cystic fibrosis carrier in an individual comprising: (a)generating an amplicon library by amplifying multiple target segments ofa CFTR nucleic acid obtained from the individual, wherein the sampleCFTR nucleic acid is extracted from a dried biological fluid sampleeluted from an absorbent tip of a microsampling device; (b) sequencingthe amplicons in the amplicon library using high throughput massiveparallel sequencing, and (c) detecting a genetic basis for beingaffected with cystic fibrosis, or for being a cystic fibrosis carrierwhen the nucleotide sequence of one or more of the target segments ofthe CFTR nucleic acid comprises a mutation associated with cysticfibrosis.

Also provided herein are kits for detecting at least one mutation in asample CFTR nucleic acid in a dried biological fluid sample comprising askin puncture tool, a volumetric absorptive microsampling device, alysis buffer, and proteinase K, wherein the at least one mutationcomprises one or more of the CFTR mutations listed in Table 2.

Additionally or alternatively, in some embodiments, the kits furthercomprise one or more primer pairs that hybridize to one or more targetsegments of the sample CFTR nucleic acid. In some embodiments, the kitsfurther comprise one or more bait sequences that hybridize to one ormore target segments of the sample CFTR nucleic acid. In someembodiments, the lysis buffer comprises guanidine hydrochloride,Tris.Cl, EDTA, Tween 20, and Triton X-100.

In any of the above embodiments of the kits of the present technology,the volumetric absorptive microsampling device is a MITRA® tip.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the DNA yield from one-tip versus dual-tipextraction from MITRA® tips using the DNA Investigator kit on theQIAsymphony® automated extraction platform. For dual-tip extraction, thelysates eluted from two individual MITRA® tips from the same patientwere combined together. FIG. 1 demonstrates that dual-tip extraction onaverage results in a 2-fold increase in DNA yield.

FIG. 2 shows the read coverage per covered CFTR target region (orhotspot) on the Cystic Fibrosis Expanded Panel (CFvantage® ExpandedScreen). FIG. 2 demonstrates that all 7 MITRA® tip specimens exceededthe minimum QC criteria for all covered CFTR target regions in a fullyburdened 384 sample run (i.e., the specimens were run on a plate thatwas performed at full capacity, i.e., 377 operations samples (Ops) and 7MITRA® tip samples).

DETAILED DESCRIPTION

The present disclosure provides methods for determining whether apatient exhibiting cystic fibrosis symptoms, or a patient at risk forcystic fibrosis, will benefit from treatment with one or moreanti-cystic fibrosis therapeutic agents. These methods are based ondetecting hereditary cystic fibrosis-related mutations in small-volumedried biological fluid samples that are collected using a volumetricabsorptive microsampling device. Kits for use in practicing the methodsare also provided.

Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present technology belongs.

As used herein, the term “about” in reference to a number is generallytaken to include numbers that fall within a range of 1%-10% in eitherdirection (greater than or less than) of the number unless otherwisestated or otherwise evident from the context.

The term “adapter” refers to a short, chemically synthesized, nucleicacid sequence which can be used to ligate to the end of a nucleic acidsequence in order to facilitate attachment to another molecule. Theadapter can be single-stranded or double-stranded. An adapter canincorporate a short (typically less than 50 base pairs) sequence usefulfor PCR amplification or sequencing.

As used herein, the “administration” of a therapeutic agent or drug to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

As used herein, an “alteration” of a gene or gene product (e.g., amarker gene or gene product) refers to the presence of a mutation ormutations within the gene or gene product, e.g., a mutation, whichaffects the quantity or activity of the gene or gene product, ascompared to the normal or wild-type gene. The genetic alteration canresult in changes in the quantity, structure, and/or activity of thegene or gene product in a diseased tissue or cell, as compared to itsquantity, structure, and/or activity, in a normal or healthy tissue orcell (e.g., a control). For example, an alteration which is associatedwith cystic fibrosis may have an altered nucleotide sequence (e.g., amutation), amino acid sequence, chromosomal translocation,intra-chromosomal inversion, copy number, expression level, proteinlevel, protein activity, of the gene encoding the membrane-associatedprotein “cystic fibrosis transmembrane regulator” (or “CFTR”) in adiseased tissue or cell, as compared to that observed within a normal,healthy tissue or cell. Exemplary mutations include, but are not limitedto, point mutations (e.g., silent, missense, or nonsense), deletions,insertions, inversions, linking mutations, duplications, translocations,inter- and intra-chromosomal rearrangements. Mutations can be present inthe coding or non-coding region of the gene. In certain embodiments, thealterations are associated with a phenotype, e.g., a phenotypeassociated with cystic fibrosis.

As used herein, the terms “amplify” or “amplification” with respect tonucleic acid sequences, refer to methods that increase therepresentation of a population of nucleic acid sequences in a sample.Copies of a particular target nucleic acid sequence generated in vitroin an amplification reaction are called “amplicons” or “amplificationproducts”. Amplification may be exponential or linear. A target nucleicacid may be DNA (such as, for example, genomic DNA and cDNA) or RNA.While the exemplary methods described hereinafter relate toamplification using polymerase chain reaction (PCR), numerous othermethods such as isothermal methods, rolling circle methods, etc., arewell known to the skilled artisan. The skilled artisan will understandthat these other methods may be used either in place of, or togetherwith, PCR methods. See, e.g., Saiki, “Amplification of Genomic DNA” inPCR Protocols, Innis et al., Eds., Academic Press, San Diego, Calif.1990, pp 13-20; Wharam, et al., Nucleic Acids Res. 29(11):E54-E54(2001).

“Bait”, as used herein, is a type of hybrid capture reagent thatretrieves target nucleic acid sequences for sequencing. A bait can be anucleic acid molecule, e.g., a DNA or RNA molecule, which can hybridizeto (e.g., be complementary to), and thereby allow capture of a targetnucleic acid. In one embodiment, a bait is an RNA molecule (e.g., anaturally-occurring or modified RNA molecule); a DNA molecule (e.g., anaturally-occurring or modified DNA molecule), or a combination thereof.In other embodiments, a bait includes a binding entity, e.g., anaffinity tag, that allows capture and separation, e.g., by binding to abinding entity, of a hybrid formed by a bait and a nucleic acidhybridized to the bait. In one embodiment, a bait is suitable forsolution phase hybridization.

As used herein, “bait set” refers to one or a plurality of baitmolecules.

The term “carrier state” or “cystic fibrosis carrier” as used hereinmeans a person who harbors a CFTR allele that has a mutant CFTR nucleicacid sequence associated with cystic fibrosis, and a second allele thatis not a mutant CFTR nucleic acid sequence. Cystic fibrosis is an“autosomal recessive” disease, meaning that a mutation produces littleor no phenotypic effect when present in a heterozygous state with anon-disease related allele, but produces a “disease state” when a personis homozygous or a compound heterozygote, i.e., both CFTR alleles aremutant CFTR nucleic acid sequences.

A “CFTR nucleic acid” as used herein refers to a nucleic acid thatcontains a sequence of a CFTR gene, mRNA, cDNA or a portion of such aCFTR sequence. A CFTR nucleic acid may contain the CFTR coding region. ACFTR nucleic acid may be genomic DNA, cDNA, single stranded DNA or mRNA.In some embodiments, only a single strand of a sample CFTR nucleic acidis amplified and/or sequenced. In some embodiments both strands ofdouble stranded CFTR DNA are amplified and sequenced. A CFTR nucleicacid may be present in a biological sample or it may be isolated from abiological sample.

The term “CFTR promoter region” as used herein refers to a segment ofthe CFTR gene representing at least the first 250 nucleotides (nt)upstream from the translation start site. In other embodiments, thepromoter region may include the first 250 nt, first 300 nt, first 350nt, first 400 nt, first 450 nt, first 500 nt, first 1 kb, first 5 kb,first 10, kb, first 15, kb, first 20, kb, first 21 kb, or first 22 kb ofsequence directly upstream of the start codon. A deletion of thepromoter region as defined herein may be accompanied by deletion ofdownstream exons/introns but not all of the CFTR gene. In someembodiments, the coordinate deletion involving the CFTR promoter regionand downstream CFTR gene sequence involves about less than 10 exons, andmore typically involves less than 5 exons. Deletions or duplications ofthe CFTR promoter region may be detected using primers that flank thedeleted or duplicated sequence. In certain embodiments, a promoterdeletion or duplication involves a segment of at least one or morenucleotides, at least four or more nucleotides, at least 5 or morenucleotides, at least 8 or more nucleotides, or at least 12 or morenucleotides.

The term “coding sequence” as used herein means a sequence of a nucleicacid or its complement, or a part thereof, that can be transcribedand/or translated to produce the corresponding mRNA, and/or polypeptideor a fragment thereof. Coding sequences include exons in a genomic DNAor immature primary RNA transcripts, which are joined together by thecell's biochemical machinery to provide a mature mRNA. The anti-sensestrand is the complement of such a nucleic acid, and the encodingsequence can be deduced there from. The term “non-coding sequence” asused herein means a sequence of a nucleic acid or its complement, or apart thereof, that is not transcribed into amino acids in vivo, or wheretRNA does not interact to place or attempt to place an amino acid.Non-coding sequences include both intron sequences in genomic DNA orimmature primary RNA transcripts, and gene-associated sequences such aspromoters, enhancers, silencers, etc.

The terms “complement”, “complementary” or “complementarity” as usedherein with reference to polynucleotides (i.e., a sequence ofnucleotides such as an oligonucleotide or a target nucleic acid) referto the Watson/Crick base-pairing rules. The complement of a nucleic acidsequence as used herein refers to an oligonucleotide which, when alignedwith the nucleic acid sequence such that the 5′ end of one sequence ispaired with the 3′ end of the other, is in “antiparallel association.”For example, the sequence “5′-A-G-T-3′” is complementary to the sequence“3′-T-C-A-S′.” Certain bases not commonly found in naturally-occurringnucleic acids may be included in the nucleic acids described herein.These include, for example, inosine, 7-deazaguanine, Locked NucleicAcids (LNA), and Peptide Nucleic Acids (PNA). Complementarity need notbe perfect; stable duplexes may contain mismatched base pairs,degenerative, or unmatched bases. Those skilled in the art of nucleicacid technology can determine duplex stability empirically considering anumber of variables including, for example, the length of theoligonucleotide, base composition and sequence of the oligonucleotide,ionic strength and incidence of mismatched base pairs. A complementsequence can also be an RNA sequence complementary to the DNA sequenceor its complement sequence, and can also be a cDNA.

The term “substantially complementary” as used herein means that twosequences hybridize under stringent hybridization conditions. Theskilled artisan will understand that substantially complementarysequences need not hybridize along their entire length. In particular,substantially complementary sequences may comprise a contiguous sequenceof bases that do not hybridize to a target sequence, positioned 3′ or 5′to a contiguous sequence of bases that hybridize under stringenthybridization conditions to a target sequence.

As used herein, a “control” or “reference” is an alternative sample usedin an experiment for comparison purpose. A control can be “positive” or“negative.” A “control nucleic acid sample” or “reference nucleic acidsample” as used herein, refers to nucleic acid molecules from a controlor reference sample. In certain embodiments, the reference or controlnucleic acid sample is a wild-type or a non-mutated DNA or RNA sequence.In certain embodiments, the reference nucleic acid sample is purified orisolated (e.g., it is removed from its natural state). In otherembodiments, the reference nucleic acid sample is from a non-diseasedsample, e.g., a blood control, a tissue control, or any othernon-diseased sample from the same or a different subject.

“Coverage depth” refers to the number of nucleotides from sequencingreads that are mapped to a given position.

The term “deletion” as used herein encompasses a mutation that removesone or more nucleotides from nucleic acid. Conversely, the term“duplication” refers to a mutation that inserts one or more nucleotidesof identical sequence directly next to this sequence in the nucleicacid. In some embodiments, a deletion or duplication involves a segmentof four or more nucleotides.

As used herein, the term “detecting” refers to determining the presenceof a mutation or alteration in a nucleic acid of interest in a sample.Detection does not require the method to provide 100% sensitivity.

The term “dosage” or “gene dosage” refers to the number of copies of agene, or portions of a gene, present in a sample.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount which results in the prevention of onset of or theamelioration in one or more symptoms associated with cystic fibrosis. Inthe context of therapeutic or prophylactic applications, the amount of atherapeutic agent administered to the subject will depend on the typeand severity of the disease and on the characteristics of theindividual, such as general health, age, sex, body weight and toleranceto drugs. It will also depend on the degree, severity and type ofdisease. The skilled artisan will be able to determine appropriatedosages depending on these and other factors. As used herein, a“therapeutically effective amount” of a therapeutic drug or agent ismeant levels in which the physiological effects of a hereditary disordersuch as cystic fibrosis are, at a minimum, ameliorated. Atherapeutically effective amount can be given in one or moreadministrations.

As used herein, the terms “extraction” or “isolation” refer to anyaction taken to separate nucleic acids from other cellular materialpresent in the sample. The term extraction or isolation includesmechanical or chemical lysis, addition of detergent or protease, orprecipitation and removal of other cellular material.

The term “flanking” as used herein with regard to primers means that aprimer hybridizes to a target nucleic acid adjoining a region ofinterest sought to be amplified on the target. The skilled artisan willunderstand that optimal primers are pairs of primers that hybridize 5′from a region of interest, one on each strand of a target doublestranded DNA molecule, such that nucleotides may be added to the 3′ endof the primer by a suitable DNA polymerase. Primers that flank a CFTRexon are generally designed not to anneal to the exon sequence butrather to anneal to sequence that adjoins the exon (e.g., intronsequence). However, in some embodiments, amplification primer may bedesigned to anneal to the exon sequence.

“Gene” as used herein refers to a DNA sequence that comprises regulatoryand coding sequences necessary for the production of an RNA, which mayhave a non-coding function (e.g., a ribosomal or transfer RNA) or whichmay include a polypeptide or a polypeptide precursor. The RNA orpolypeptide may be encoded by a full length coding sequence or by anyportion of the coding sequence so long as the desired activity orfunction is retained. Although a sequence of the nucleic acids may beshown in the form of DNA, a person of ordinary skill in the artrecognizes that the corresponding RNA sequence will have a similarsequence with the thymine being replaced by uracil, i.e., “T” isreplaced with “U.”

A “genetic basis for cystic fibrosis” in an individual refers to theindividual's genotype, in particular, of their CFTR nucleic acids andwhether the individual possesses at least one CFTR mutation thatcontributes to cystic fibrosis. The term “wild-type” as used herein withrespect to the CFTR gene or a locus thereof refers to the CFTR genesequence which is found in NCBI GenBank locus IDs M58478 (HUMCFTC),AC000111 and AC000061. A cDNA for a CFTR gene may be found in Audrezetet al., Hum. Mutat. 23(4), 343-357 (2004) and/or Genbank accessionnumber NM_000492.3. A “rare CFTR mutation” is a mutation in the CFTRgene sequence that is present in <0.1% of cystic fibrosis patients. A“private CFTR mutation” is a mutation in the CFTR gene sequence that isonly found in a single family or a small group. A “common CFTR mutation”is a mutation in the CFTR gene sequence that is associated with cysticfibrosis and is present in at least 0.1% of patients with cysticfibrosis.

The term “hybridize” as used herein refers to a process where twosubstantially complementary nucleic acid strands (at least about 65%complementary over a stretch of at least 14 to 25 nucleotides, at leastabout 75%, or at least about 90% complementary) anneal to each otherunder appropriately stringent conditions to form a duplex orheteroduplex through formation of hydrogen bonds between complementarybase pairs. Hybridizations are typically and preferably conducted withprobe-length nucleic acid molecules, preferably 15-100 nucleotides inlength, more preferably 18-50 nucleotides in length. Nucleic acidhybridization techniques are well known in the art. See, e.g., Sambrook,et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Press, Plainview, N.Y. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is influenced by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, and the thermal melting point (T_(m)) of the formed hybrid.Those skilled in the art understand how to estimate and adjust thestringency of hybridization conditions such that sequences having atleast a desired level of complementarity will stably hybridize, whilethose having lower complementarity will not. For examples ofhybridization conditions and parameters, see, e.g., Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994,Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus,N.J. In some embodiments, specific hybridization occurs under stringenthybridization conditions. An oligonucleotide or polynucleotide (e.g., aprobe or a primer) that is specific for a target nucleic acid will“hybridize” to the target nucleic acid under suitable conditions.

As used herein, the terms “individual”, “patient”, or “subject” can bean individual organism, a vertebrate, a mammal, or a human. In apreferred embodiment, the individual, patient or subject is a human.

As used herein, the term “library” refers to a collection of nucleicacid sequences, e.g., a collection of nucleic acids derived from wholegenomic, subgenomic fragments, cDNA, cDNA fragments, RNA, RNA fragments,or a combination thereof. In one embodiment, a portion or all of thelibrary nucleic acid sequences comprises an adapter sequence. Theadapter sequence can be located at one or both ends. The adaptersequence can be useful, e.g., for a sequencing method (e.g., an NGSmethod), for amplification, for reverse transcription, or for cloninginto a vector.

The library can comprise a collection of nucleic acid sequences, e.g., atarget nucleic acid sequence (e.g., a CFTR nucleic acid sequence), areference nucleic acid sequence, or a combination thereof). In someembodiments, the nucleic acid sequences of the library can be derivedfrom a single subject. In other embodiments, a library can comprisenucleic acid sequences from more than one subject (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30 or more subjects). In some embodiments, two or morelibraries from different subjects can be combined to form a libraryhaving nucleic acid sequences from more than one subject. In oneembodiment, the subject is a human having, or at risk of having, acystic fibrosis.

A “library nucleic acid sequence” refers to a nucleic acid molecule,e.g., a DNA, RNA, or a combination thereof, that is a member of alibrary. Typically, a library nucleic acid sequence is a DNA molecule,e.g., genomic DNA or cDNA. In some embodiments, a library nucleic acidsequence is fragmented, e.g., sheared or enzymatically prepared, genomicDNA. In certain embodiments, the library nucleic acid sequences comprisesequence from a subject and sequence not derived from the subject, e.g.,adapter sequence, a primer sequence, or other sequences that allow foridentification, e.g., “barcode” sequences. In some embodiments, thelibrary comprises amplicons corresponding multiple segments of a targetnucleic acid sequence, such as a sample CFTR nucleic acid sequence.

The term “multiplex PCR” as used herein refers to amplification of twoor more products which are each primed using a distinct primer pair.

“Next generation sequencing or NGS” as used herein, refers to anysequencing method that determines the nucleotide sequence of eitherindividual nucleic acid molecules (e.g., in single molecule sequencing)or clonally expanded proxies for individual nucleic acid molecules in ahigh throughput parallel fashion (e.g., greater than 10³, 10⁴, 10⁵ ormore molecules are sequenced simultaneously). In one embodiment, therelative abundance of the nucleic acid species in the library can beestimated by counting the relative number of occurrences of theircognate sequences in the data generated by the sequencing experiment.Next generation sequencing methods are known in the art, and aredescribed, e.g., in Metzker, M. Nature Biotechnology Reviews 11:31-46(2010).

As used herein, “oligonucleotide” refers to a molecule that has asequence of nucleic acid bases on a backbone comprised mainly ofidentical monomer units at defined intervals. The bases are arranged onthe backbone in such a way that they can bind with a nucleic acid havinga sequence of bases that are complementary to the bases of theoligonucleotide. The most common oligonucleotides have a backbone ofsugar phosphate units. A distinction may be made betweenoligodeoxyribonucleotides that do not have a hydroxyl group at the 2′position and oligoribonucleotides that have a hydroxyl group at the 2′position. Oligonucleotides may also include derivatives, in which thehydrogen of the hydroxyl group is replaced with organic groups, e.g., anallyl group. Oligonucleotides that function as primers or probes aregenerally at least about 10-15 nucleotides in length, or up to about 70,100, 110, 150 or 200 nucleotides in length, and more preferably at leastabout 15 to 25 nucleotides in length. Oligonucleotides used as primersor probes for specifically amplifying or specifically detecting aparticular target nucleic acid generally are capable of specificallyhybridizing to the target nucleic acid.

As used herein, the term “primer” refers to an oligonucleotide, which iscapable of acting as a point of initiation of nucleic acid sequencesynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a target nucleic acid strandis induced, i.e., in the presence of different nucleotide triphosphatesand a polymerase in an appropriate buffer (“buffer” includes pH, ionicstrength, cofactors etc.) and at a suitable temperature. One or more ofthe nucleotides of the primer can be modified for instance by additionof a methyl group, a biotin or digoxigenin moiety, a fluorescent tag orby using radioactive nucleotides. A primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being substantially complementaryto the strand. The term primer as used herein includes all forms ofprimers that may be synthesized including peptide nucleic acid primers,locked nucleic acid primers, phosphorothioate modified primers, labeledprimers, and the like. The term “forward primer” as used herein means aprimer that anneals to the anti-sense strand of double-stranded DNA(dsDNA). A “reverse primer” anneals to the sense-strand of dsDNA.

Primers are typically at least 10, 15, 18, or 30 nucleotides in lengthor up to about 100, 110, 125, or 200 nucleotides in length. In someembodiments, primers are preferably between about 15 to about 60nucleotides in length, and most preferably between about 25 to about 40nucleotides in length. In some embodiments, primers are 15 to 35nucleotides in length. There is no standard length for optimalhybridization or polymerase chain reaction amplification. An optimallength for a particular primer application may be readily determined inthe manner described in H. Erlich, PCR Technology, PRINCIPLES ANDAPPLICATION FOR DNA AMPLIFICATION, (1989).

As used herein, the term “primer pair” refers to a forward and reverseprimer pair (i.e., a left and right primer pair) that can be usedtogether to amplify a given region of a nucleic acid of interest.

“Probe” as used herein refers to a nucleic acid that interacts with atarget nucleic acid via hybridization. A probe may be fullycomplementary to a target nucleic acid sequence or partiallycomplementary. The level of complementarity will depend on many factorsbased, in general, on the function of the probe. Probes can be labeledor unlabeled, or modified in any of a number of ways well known in theart. A probe may specifically hybridize to a target nucleic acid. Probesmay be DNA, RNA or a RNA/DNA hybrid. Probes may be oligonucleotides,artificial chromosomes, fragmented artificial chromosome, genomicnucleic acid, fragmented genomic nucleic acid, RNA, recombinant nucleicacid, fragmented recombinant nucleic acid, peptide nucleic acid (PNA),locked nucleic acid, oligomer of cyclic heterocycles, or conjugates ofnucleic acid. Probes may comprise modified nucleobases, modified sugarmoieties, and modified internucleotide linkages. Probes are typically atleast about 10, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100 nucleotides ormore in length.

As used herein, the term “sample” refers to clinical samples obtainedfrom a patient. In preferred embodiments, a sample is obtained from abiological source (i.e., a “biological sample”), such as tissue orbodily fluid collected from a subject. Sample sources include, but arenot limited to, mucus, sputum (processed or unprocessed), bronchialalveolar lavage (BAL), bronchial wash (BW), blood, bodily fluids,cerebrospinal fluid (CSF), urine, plasma, serum, or tissue (e.g., biopsymaterial). Preferred sample sources include plasma, serum, or wholeblood.

A “sample CFTR nucleic acid” is a CFTR nucleic acid in, or isolatedfrom, a biological sample. Processing methods to release or otherwisemake available a nucleic acid for detection are well known in the artand may include steps of nucleic acid manipulation, e.g., DNA or RNAextraction from a biological sample, and preparing a cDNA by reversetranscription of RNA from the biological sample. A biological sample maybe a body fluid or a tissue sample. In some embodiments, a biologicalsample may comprise blood, plasma, sera, urine, feces, epidermal sample,vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, culturedcells, bone marrow sample and/or chorionic villi, cultured cells, andthe like. In some embodiments, the biological sample may be a driedbiological fluid sample collected by a volumetric absorptivemicrosampling device. Fixed or frozen tissues also may be used. Amnioticfluid of 10-15 ml, cultured cells which are 80-100% confluent in twoT-25 flasks and 25 mg of chorionic villi are useful sample amounts forprocessing.

The term “sensitivity,” as used herein in reference to the methods ofthe present technology, is a measure of the ability of a method todetect a preselected sequence variant in a heterogeneous population ofsequences. A method has a sensitivity of S % for variants of F % if,given a sample in which the preselected sequence variant is present asat least F % of the sequences in the sample, the method can detect thepreselected sequence at a preselected confidence of C %, S % of thetime. By way of example, a method has a sensitivity of 90% for variantsof 5% if, given a sample in which the preselected variant sequence ispresent as at least 5% of the sequences in the sample, the method candetect the preselected sequence at a preselected confidence of 99%, 9out of 10 times (F=5%; C=99%; S=90%). Exemplary sensitivities include atleast 50, 60, 70, 80, 90, 95, 98, and 99%.

“Sequencing depth” or “read depth” as used herein refers to the numberof times a sequence has been sequenced (the depth of sequencing). As anexample, read depth can be determined by aligning multiple sequencingrun results and counting the start position of reads in nonoverlappingwindows of a certain size (for example, 100 bp). Copy number variationcan be determined based on read depth using methods known in the art,for example, the methods described in Yoon et al., Genome ResearchSeptember; 19(9):1586-1592 (2009); Xie et al., BMC Bioinformatics 10:80(2009); or Medvedev et al., Nature Methods 6(11 Suppl):513-20 (2009).Use of this type of method and analysis is referred to as a “read depthapproach.”

The term “specific” as used herein in reference to an oligonucleotideprimer means that the nucleotide sequence of the primer has at least 12bases of sequence identity with a portion of the nucleic acid to beamplified when the oligonucleotide and the nucleic acid are aligned. Anoligonucleotide primer that is specific for a nucleic acid is one that,under the stringent hybridization or washing conditions, is capable ofhybridizing to the target of interest and not substantially hybridizingto nucleic acids which are not of interest. Higher levels of sequenceidentity are preferred and include at least 75%, at least 80%, at least85%, at least 90%, at least 85-95%, and more preferably at least 98%sequence identity. Sequence identity can be determined using acommercially available computer program with a default setting thatemploys algorithms well known in the art. As used herein, sequences thathave “high sequence identity” have identical nucleotides at least atabout 50% of aligned nucleotide positions, preferably at least at about60% of aligned nucleotide positions, and more preferably at least atabout 75% of aligned nucleotide positions.

“Specificity,” as used herein, is a measure of the ability of a methodto distinguish a truly occurring preselected sequence variant fromsequencing artifacts or other closely related sequences. It is theability to avoid false positive detections. False positive detectionscan arise from errors introduced into the sequence of interest duringsample preparation, sequencing error, or inadvertent sequencing ofclosely related sequences like pseudo-genes or members of a gene family.A method has a specificity of X % if, when applied to a sample set ofN_(Total) sequences, in which X_(True) sequences are truly variant andX_(Not true) are not truly variant, the method selects at least X % ofthe not truly variant as not variant. E.g., a method has a specificityof 90% if, when applied to a sample set of 1,000 sequences, in which 500sequences are truly variant and 500 are not truly variant, the methodselects 90% of the 500 not truly variant sequences as not variant.Exemplary specificities include at least 50, 60, 70, 80, 90, 95, 98, and99%.

The term “stringent hybridization conditions” as used herein refers tohybridization conditions at least as stringent as the following:hybridization in 50% formamide, 5×SSC, 50 mM NaH₂PO4, pH 6.8, 0.5% SDS,0.1 mg/mL sonicated salmon sperm DNA, and 5×Denhart's solution at 42° C.overnight; washing with 2×SSC, 0.1% SDS at 45° C.; and washing with0.2×SSC, 0.1% SDS at 45° C. In another example, stringent hybridizationconditions should not allow for hybridization of two nucleic acids whichdiffer over a stretch of 20 contiguous nucleotides by more than twobases.

The terms “target nucleic acid” or “target sequence” or “target segment”as used herein refer to a nucleic acid sequence of interest to bedetected and/or quantified in the sample to be analyzed. Target nucleicacid may be composed of segments of a chromosome, a complete gene withor without intergenic sequence, segments or portions of a gene with orwithout intergenic sequence, or sequence of nucleic acids which probesor primers are designed. Target nucleic acids may include a wild-typesequence(s), a mutation, deletion, insertion or duplication, tandemrepeat elements, a gene of interest, a region of a gene of interest orany upstream or downstream region thereof. Target nucleic acids mayrepresent alternative sequences or alleles of a particular gene. Targetnucleic acids may be derived from genomic DNA, cDNA, or RNA.

As used herein, the terms “treat,” “treating” or “treatment” refer, toan action to obtain a beneficial or desired clinical result including,but not limited to, alleviation or amelioration of one or more signs orsymptoms of a disease or condition (e.g., regression, partial orcomplete), diminishing the extent of disease, stability (i.e., notworsening, achieving stable disease) state of disease, amelioration orpalliation of the disease state, diminishing rate of or time toprogression, and remission (whether partial or total).

Microsampling Devices Employed in the Methods of the Present Technology

Conventional dried blood spotting techniques are accompanied by a numberof drawbacks, including imprecise sample volume and reliance on aconstant sample viscosity (i.e., the expectation that the sample willspread uniformly on the sample card). A constant viscosity results inblood spot diameters remaining constant when equal volume samples areadministered to the cards. However, viscosity varies significantlybetween blood samples because of differing hematocrit (HCT) or packedcell volume (PCV) levels in the blood. Samples with high hematocritlevels form smaller diameter spots on the bloodspot papers, leading todifferent concentrations of blood within the fixed diameter of the spotssampled. PCV levels are believed to show a variance of about 45% in spotdiameters. As internal standards are sprayed onto the spotted blood,this can result in a 45% error in quantitation. The microsamplingdevices employed in the methods disclosed herein confer severaladvantages, including the collection of more precise blood volumes, lackof hematocrit bias, and the ability to be easily automated with standardliquid handlers for lab processing.

Additionally, conventional blood spot techniques require a comparativelylarge volume of blood relative to the disclosed microsampling devices. Adried blood spot would generally require 50-75 μl per spot, while amicrosampling device can yield results from approximately 20 μl. It hasbeen recognized in the art that dried blood spots often have performancevariability issues for detecting viral load compared to other samplestypes, such as plasma (Pannus et al., Medicine, 95:48(e5475) (2016)),and the volume of a dried blood spot may need to be significantly higherfor certain types of assessment (e.g., optical density) compared toother sample types, such as serum (Brandao et al., J. Clin. Virol.,57:98-102 (2013)). Indeed, found that using both dried blood spot andplasma spot screening for detecting viral load and treatment failure inHIV patients receiving antiretroviral therapy found that both yielded ahigh rate of false positives (Sawadogo et al., J. Clin. Microbiol.,52(11):3878-83 (2014)).

The microsampling device useful in the methods of the present technologycomprises an absorbent tip having a distal end and a proximal end. Thewidth of the distal end of the absorbent tip is narrow compared to thewidth of the proximal end. The proximal end is attached to a holder,whereas the distal end is configured to contact a fluid to be absorbed,such as blood. The microsampling device permits biological fluidsamples, such as blood, to be easily dried, shipped, and then lateranalyzed. In certain embodiments, the biological fluid is blood from afingerstick.

Wicking action draws the blood into the absorbent tip. An optionalbarrier between the absorbent tip and the holder prevents blood frompassing or wicking to the holder. The absorbent tip is composed of amaterial that wicks up substantially the same volume of fluid even whenexcess fluid is available (volumetric absorptive microsampling orVAMS™). The volume of the absorbent tip affects the volume of fluidabsorbed. The size and shape of the absorbent tip may be varied toadjust the volume of absorbed blood and the rate of absorption. Bloodvolumes of about 7-15 μL, about 20 μL, and even up to about 30 μL may beacceptable. The sampling time may be about 2 seconds, about 3 seconds,about 5 seconds, or up to about 10 seconds.

In some embodiments, the material used for the absorbent tip ishydrophilic (e.g., polyester). Alternatively, the material may initiallybe hydrophobic and is subsequently treated to make it hydrophilic.Hydrophobic matrices may be rendered hydrophilic by a variety of knownmethods, such as plasma treatment or surfactant treatment (e.g.,Tween-40 or Tween-80) of the matrix. In some embodiments, plasmatreatment is used to render a hydrophobic material such as polyolefin,e.g., polyethylene, hydrophilic. Alternatively, the grafting ofhydrophilic polymers to the surface and the chemical functionalizationof active groups on the surface with polar or hydrophilic molecules suchas sugars can be used to achieve a hydrophilic surface for the absorbenttip. Covalent modification could also be used to add polar orhydrophilic functional groups to the surface of absorbent tip. Othersuitable materials for the absorbent tip include sintered glass,sintered steel, sintered ceramics, and sintered polymers of plastic, andsintered polyethylene.

In some embodiments, the microsampling device comprises an absorbent tipmade of a hydrophilic polymeric material of sufficient size to absorb amaximum of about 20 μL of blood in about 2-5 seconds, and having alength of less than about 5 mm (0.2 inches) and a cross-sectional areaof less than about 20 mm² and a density of less than about 4 g/cc. Insome embodiments, the absorbent tips are composed of polyethylene andconfigured to absorb about 1-20 microliters of blood, preferably within1-7 seconds, and more preferably within about 1-5 seconds. The absorbenttip may contain one or more of dried blood, dried anticoagulant or aninternal standard.

In certain embodiments, the absorbent tips have a volume of about 35mm³, absorb about 13-14 microliters of blood in about 3 seconds, absorb9-10 microliters of blood in about 2.5 seconds, and have a pore volumeof about 38%. In other embodiments, the absorbent tips have a volume ofabout 24 microliters, a density of about 0.6 g/cc, absorb about 10microliters of blood in about 2.5 seconds, and have a pore volume ofabout 40%. In some embodiments, the volumetric absorptive microsamplingdevice is a MITRA® tip, as described in US 2013/0116597, which is hereinincorporated by reference in its entirety.

The absorbent tip may be shaped with an exterior resembling a truncatedcone with a narrow and rounded distal end. In some embodiments, theholder has a cylindrical post that fits into a recess inside the centerof the absorbent tip and extending along the longitudinal axis of theabsorbent tip and holder. The conical shape of the absorbent tip helpswick the sample quickly and uniformly.

The holder may be adapted for use with a pipette. In some embodiments, atubular, conical shaped holder is preferred, with the absorbent tip onthe narrow end of the holder. The wider opposite end of the holder maybe closed, or open and hollow, and may optionally be configured toattach to a pipette tip. The holder may have outwardly extending flangesthat are arranged to abut mating structures in holders, drying racks ortest equipment to help position the absorbent tip at desired locationsin such holders, drying racks and test equipment.

In certain embodiments, the holder may include a pipette tip or atapering, tubular structure configured to nest with a pipette tip. Theabsorbent tip may be composed of polyethylene, and both the absorbenttip and holder are made under aseptic conditions, or are terminallysterilized. The absorbent tip may contain dried anti-coagulant. In someembodiments, the holder has a plurality of ribs extending along a lengthof the holder. The ribs may have a height and length selected to keepthe absorbent tip from contacting walls of a recess into which theholder and absorbent tip are placed for shipment, or for extraction ofthe dried blood in the absorbent tip.

After absorbing a small-volume sample, the absorbent tip is then dried.In some embodiments, the small-volume blood sample is dried for at least10 minutes, at least 20 minutes, at least 30 minutes, at least 40minutes, at least 50 minutes, at least 1 hour, at least 2 hours, atleast 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, atleast 8 hours, at least 12 hours, at least 16 hours, at least 20 hours,at least 24 hours, at least 48 hours, at least 72 hours, or at least 96hours at ambient or room temperature. In certain embodiments, thesmall-volume blood sample is dried for about 2-3 hours.

Drying can be done on a suitable rack or holder, or preferably theabsorbent tip and holder can be transferred to a special dryingcontainer configured to facilitate drying while minimizing contactbetween the absorbent tip and the walls of the drying container or otherpotential contaminant surfaces. The drying container may have adesiccant to facilitate drying. The drying container may also provide aprotective cover which may be sealed for transport to preventcontamination. In some embodiments, the cover has a surface onto whichprinted indicia may be written to identify the source of the dried bloodsample and provide other relevant information. In some embodiments, thedimensions of the container, and the relative positions of the holderswithin the container, will conform to SBS Microwell platespecifications. The microsampling device and the drying container may beplaced in a plastic bag along with a desiccant to assist with drying andcan either be shipped in this fashion, or shipped after the desiccant isremoved.

In some embodiments, the wider opposite end of the holder is hollow andthe container has a first portion with a mounting projection portionsized to fit into and releasably engage the hollow end of the holder.Additionally or alternatively, the container has a second portionreleasably fastened to the first portion and has a recess configured toenclose a portion of the holder for transportation of the holder. Thecontainer may comprise a plurality of openings allowing air to accessthe absorbent tip of the microsampling device. Moreover, the firstportion may have a side with an access port therein of sufficient sizeand located so that indicia may be applied through the port and onto theholder when the holder is on the mounting projection.

Upon receipt at the testing location, the absorbent tip may be eluted ina predetermined volume of a suitable buffer (as described herein) eithermanually or via automated means to extract the nucleic acids or proteinsof interest from dried blood. Physical agitation techniques such assonication or vortexing of the fluid and/or the absorbent tip mayaccelerate the extraction process from the dried blood into a liquidsample matrix. Physical separation techniques such as centrifugation,evaporation/reconstitution, concentration, precipitation, liquid/liquidextraction, and solid phase extraction can be used to further simplifythe sample matrix for further analysis.

Each container may enclose a plurality of holders, wherein each holdercomprises an absorbent tip at its distal end and has a hollow proximalend. The container likewise has a plurality of elongated mountingprojections each sized to fit into and releasably engage the hollow endsof the plurality of holders. The second portion of the container hasrecesses configured to separately enclose each of the plurality ofholders in a separate enclosure within the container. In certainembodiments, each of the plurality of holders has a plurality of ribsextending along a length of the holder with the ribs configured to keepthe absorbent tip from contacting walls of the container. As desired, adesiccant may be placed inside the container to help dry the blood inthe absorbent tip or maintain dryness. Each holder may have visibleindicia associating the holder with the container and with at least oneother holder, such as serial numbers with various portions of the numberindicating related holders/absorbent tips and the container in which theholders are shipped.

Nucleic Acid Extraction

In one aspect, the present disclosure provides a method for extractinggenomic DNA from a dried biological fluid sample collected with avolumetric absorptive microsampling device (e.g., MITRA® Tip). In someembodiments, the dried biological fluid sample is eluted by contactingthe absorbent tip of a volumetric absorptive microsampling device with alysis buffer and proteinase K. The lysis buffer may comprise guanidinehydrochloride, Tris.Cl, EDTA, Tween 20, and Triton X-100. Proteinase Kis a broad spectrum serine protease that is stable over a wide pH range(4-12), with a pH optimum of pH 8.0. The predominant site of ProteinaseK cleavage is the peptide bond adjacent to the carboxyl group ofaliphatic and aromatic amino acids with blocked alpha amino groups.Elevating the reaction temperature from 37° C. to 50-60° C. may increasethe Proteinase K activity by several fold. Proteinase K activity can beenhanced by the addition of 0.5-1% sodium dodecyl sulfate (SDS), 3 MGuanidinium chloride, 1 M Guanidinium thiocyanate, or 4 M urea.

Alternatively, other protocols for nucleic acid extraction may be usedin the methods of the present technology. Examples of other commerciallyavailable nucleic acid purification kits include Molzym GmbH & Co KG(Bremen, D E), Qiagen (Hilden, D E), Macherey-Nagel (Düren, D E), Roche(Basel, C H) or Sigma (Deisenhofen, D E). Other systems for nucleic acidpurification, which are based on the use of polystyrene beads etc., assupport material may also be used.

In some embodiments, extraction of genomic DNA from a dried biologicalfluid sample collected with a volumetric absorptive microsampling devicecomprises denaturing nucleoprotein complexes in cells present in thedried biological fluid sample. In certain embodiments, extraction ofgenomic DNA from a dried biological fluid sample collected with avolumetric absorptive microsampling device comprises removing proteincontaminants, inactivating nuclease activity, and/or removing biologicaland/or chemical contaminants present in the dried biological fluidsample.

In some embodiments, extraction of genomic DNA from a dried biologicalfluid sample collected with a volumetric absorptive microsampling devicemay be performed using automated DNA extraction platforms. In someembodiments, the automated DNA extraction platform has high-throughputcapacity, such as up to 100 extractions per cycle. In certainembodiments, extraction of genomic DNA from a dried biological fluidsample collected with a volumetric absorptive microsampling device maybe performed using commercially available automated workstations, suchas the QIAsymphony® or Hamilton® automation. In some embodiments,extraction of genomic DNA from a dried biological fluid sample collectedwith a volumetric absorptive microsampling device is performed on an EZ1Advanced XL, EZ1 Advanced, or Biorobot® EZ1™ automated system with anEZ1 DNA Investigator Kit. In some embodiments, extraction of genomic DNAfrom a dried biological fluid sample collected with a volumetricabsorptive microsampling device is performed using commerciallyavailable reagent kits.

NGS Platforms

In some embodiments, high throughput, massively parallel sequencingemploys sequencing-by-synthesis with reversible dye terminators. Inother embodiments, sequencing is performed via sequencing-by-ligation.In yet other embodiments, sequencing is single molecule sequencing.Examples of Next Generation Sequencing techniques include, but are notlimited to pyrosequencing, Reversible dye-terminator sequencing, SOLiDsequencing, Ion semiconductor sequencing, Helioscope single moleculesequencing etc.

The Ion Torrent™ (Life Technologies, Carlsbad, Calif.) ampliconsequencing system employs a flow-based approach that detects pH changescaused by the release of hydrogen ions during incorporation ofunmodified nucleotides in DNA replication. For use with this system, asequencing library is initially produced by generating DNA fragmentsflanked by sequencing adapters. In some embodiments, these fragments canbe clonally amplified on particles by emulsion PCR. The particles withthe amplified template are then placed in a silicon semiconductorsequencing chip. During replication, the chip is flooded with onenucleotide after another, and if a nucleotide complements the DNAmolecule in a particular microwell of the chip, then it will beincorporated. A proton is naturally released when a nucleotide isincorporated by the polymerase in the DNA molecule, resulting in adetectable local change of pH. The pH of the solution then changes inthat well and is detected by the ion sensor. If homopolymer repeats arepresent in the template sequence, multiple nucleotides will beincorporated in a single cycle. This leads to a corresponding number ofreleased hydrogens and a proportionally higher electronic signal.

The 454TM GS FLX™ sequencing system (Roche, Germany), employs alight-based detection methodology in a large-scale parallelpyrosequencing system. Pyrosequencing uses DNA polymerization, addingone nucleotide species at a time and detecting and quantifying thenumber of nucleotides added to a given location through the lightemitted by the release of attached pyrophosphates. For use with the 454™system, adapter-ligated DNA fragments are fixed to small DNA-capturebeads in a water-in-oil emulsion and amplified by PCR (emulsion PCR).Each DNA-bound bead is placed into a well on a picotiter plate andsequencing reagents are delivered across the wells of the plate. Thefour DNA nucleotides are added sequentially in a fixed order across thepicotiter plate device during a sequencing run. During the nucleotideflow, millions of copies of DNA bound to each of the beads are sequencedin parallel. When a nucleotide complementary to the template strand isadded to a well, the nucleotide is incorporated onto the existing DNAstrand, generating a light signal that is recorded by a CCD camera inthe instrument.

Sequencing technology based on reversible dye-terminators: DNA moleculesare first attached to primers on a slide and amplified so that localclonal colonies are formed. Four types of reversible terminator bases(RT-bases) are added, and non-incorporated nucleotides are washed away.Unlike pyrosequencing, the DNA can only be extended one nucleotide at atime. A camera takes images of the fluorescently labeled nucleotides,then the dye along with the terminal 3′ blocker is chemically removedfrom the DNA, allowing the next cycle.

Helicos's single-molecule sequencing uses DNA fragments with added polyAtail adapters, which are attached to the flow cell surface. At eachcycle, DNA polymerase and a single species of fluorescently labelednucleotide are added, resulting in template-dependent extension of thesurface-immobilized primer-template duplexes. The reads are performed bythe Helioscope sequencer. After acquisition of images tiling the fullarray, chemical cleavage and release of the fluorescent label permitsthe subsequent cycle of extension and imaging.

Sequencing by synthesis (SBS), like the “old style” dye-terminationelectrophoretic sequencing, relies on incorporation of nucleotides by aDNA polymerase to determine the base sequence. A DNA library withaffixed adapters is denatured into single strands and grafted to a flowcell, followed by bridge amplification to form a high-density array ofspots onto a glass chip. Reversible terminator methods use reversibleversions of dye-terminators, adding one nucleotide at a time, detectingfluorescence at each position by repeated removal of the blocking groupto allow polymerization of another nucleotide. The signal of nucleotideincorporation can vary with fluorescently labeled nucleotides,phosphate-driven light reactions and hydrogen ion sensing having allbeen used. Examples of SBS platforms include Illumina GA, HiSeq 2500,HiSeq 1500, HiSeq 2000, or HiSeq 1000. The MiSeq® personal sequencingsystem (Illumina, Inc.) also employs sequencing by synthesis withreversible terminator chemistry.

In contrast to the sequencing by synthesis method, the sequencing byligation method uses a DNA ligase to determine the target sequence. Thissequencing method relies on enzymatic ligation of oligonucleotides thatare adjacent through local complementarity on a template DNA strand.This technology employs a partition of all possible oligonucleotides ofa fixed length, labeled according to the sequenced position.Oligonucleotides are annealed and ligated and the preferential ligationby DNA ligase for matching sequences results in a dinucleotide encodedcolor space signal at that position (through the release of afluorescently labeled probe that corresponds to a known nucleotide at aknown position along the oligo). This method is primarily used by LifeTechnologies' SOLiD™ sequencers. Before sequencing, the DNA is amplifiedby emulsion PCR. The resulting beads, each containing only copies of thesame DNA molecule, are deposited on a solid planar substrate.

SMRT™ sequencing is based on the sequencing by synthesis approach. TheDNA is synthesized in zero-mode wave-guides (ZMWs)-small well-likecontainers with the capturing tools located at the bottom of the well.The sequencing is performed with use of unmodified polymerase (attachedto the ZMW bottom) and fluorescently labeled nucleotides flowing freelyin the solution. The wells are constructed in a way that only thefluorescence occurring at the bottom of the well is detected. Thefluorescent label is detached from the nucleotide at its incorporationinto the DNA strand, leaving an unmodified DNA strand.

High-throughput sequencing of DNA can also take place using AnyDot-chips(Genovoxx, Germany), which allows monitoring of biological processes(e.g., miRNA expression or allele variability (SNP detection)). Forexample, the AnyDot-chips allow for 10×-50× enhancement of nucleotidefluorescence signal detection. Other high-throughput sequencing systemsinclude those disclosed in Venter, J., et al., Science 16 Feb. 2001;Adams, M. et al., Science 24 Mar. 2000; and M. J, Levene, et al.,Science 299:682-686, January 2003; as well as U.S. Application Pub. No.2003/0044781 and 2006/0078937.

Cystic Fibrosis Detection Assays of the Present Technology

Provided herein are methods for detecting at least one mutation in asample CFTR nucleic acid comprising generating a library comprisingamplicons corresponding to a plurality of target segments of the sampleCFTR nucleic acid, wherein the sample CFTR nucleic acid is extractedfrom a dried biological fluid sample eluted from an absorbent tip of amicrosampling device (e.g., MITRA® Tip) with a lysis buffer andProteinase K. In some embodiments, the at least one mutation in thesample CFTR nucleic acid is selected from among a base change, a genedeletion and a gene duplication. In certain embodiments, the at leastone mutation in the sample CFTR nucleic acid is associated with cysticfibrosis, and comprises one or more of the mutations listed in Table 2of the present disclosure. In some embodiments, the at least onemutation of the sample CFTR nucleic acid is detected using highthroughput massive parallel sequencing. In some embodiments, the lysisbuffer comprises guanidine hydrochloride, Tris.Cl, EDTA, Tween 20, andTriton X-100.

In some embodiments, the dried biological fluid sample is dried plasma,dried serum, or dried whole blood. In certain embodiments, the driedbiological fluid sample is obtained from a patient exhibiting cysticfibrosis symptoms, or has a family history of cystic fibrosis or a CFTRmutation. In some embodiments, the dried biological fluid sample isobtained from a male partner of an obstetrics and gynecology patienthaving cystic fibrosis or at least one CFTR mutation. In someembodiments, the dried biological fluid sample is obtained from anpatient who is unable to provide large volumes of blood for recurrenttesting, such as an infant or an elderly person.

In some embodiments, the dried biological fluid sample on the absorbenttip of the microsampling device is collected from a patient viafingerstick. In certain embodiments, the microsampling device is aMITRA® tip. Elution of the dried biological fluid sample may beperformed by contacting the absorbent tip of the microsampling devicewith a lysis buffer and Proteinase K. In certain embodiments, the lysisbuffer comprises guanidine hydrochloride, Tris.Cl, EDTA, Tween 20, andTriton X-100. In a further embodiment, the lysis buffer comprises 800 mMguanidine hydrochloride; 30 mM Tris.Cl, pH 8.0; 30 mM EDTA, pH 8.0; 5%Tween 20; and 0.5% Triton X-100.

Additionally or alternatively, in some embodiments, elution of the driedbiological fluid sample is performed by contacting the absorbent tip ofthe microsampling device with the lysis buffer for up to 15 minutes at90° C. Additionally or alternatively, in certain embodiments, elution ofthe dried biological fluid sample is performed by contacting theabsorbent tip of the microsampling device with Proteinase K for up to 1hour at 56° C. In other embodiments, elution of the dried biologicalfluid sample is performed by contacting the absorbent tip of themicrosampling device with Proteinase K for up to 16-18 hours at 56° C.In some embodiments, the sample volume of the microsampling device is nomore than 10-20 μL.

In some embodiments of the method, no more than 400 ng of genomic DNA iseluted from the absorbent tip of the microsampling device. In otherembodiments of the method, about 100 ng to about 400 ng of genomic DNAis eluted from the absorbent tip of the microsampling device. In someembodiments, the method further comprises ligating an adapter sequenceto the ends of the plurality of amplicons. The adapter sequence may be aP5 adapter, P7 adapter, P1 adapter, A adapter, or Ion Xpress™ barcodeadapter. Additionally or alternatively, in some embodiments, the methodfurther comprises hybridizing one or more bait sequences to one or moretarget segments of the sample CFTR nucleic acid.

In certain embodiments, the CFTR target segment that is amplified andsequenced according to the present technology may represent one or moreindividual exon(s) or portion(s) of exon(s) of the CFTR gene or one ormore portions of a CFTR mRNA or cDNA. A target segment also may includethe CFTR promoter region and/or one or more CFTR introns. In someembodiments, the target segments represent the entire CFTR gene or theentire CFTR coding region. In some embodiments, the target segmentsrepresent the entire CFTR coding region and at least one intron or aportion thereof, and an adjacent region located immediately upstream (inthe 5′ direction) of the coding sequence. The adjacent, upstream regionmay comprise from about 100 nucleotides up to about 500, 750, 1000,1100, or 1200 nucleotides of the sequence located immediately upstreamof the CFTR coding sequence. In some embodiments, the adjacent, upstreamregion comprises all or a portion of the CFTR promoter sequence. In someembodiments, the sample CFTR nucleic acid is genomic DNA.

In another aspect, the present disclosure provides a method fordetecting at least one mutation in a sample CFTR nucleic acid comprisinggenerating a library comprising amplicons corresponding to a pluralityof target segments of the sample CFTR nucleic acid, wherein the sampleCFTR nucleic acid is extracted from a dried biological fluid sampleeluted from an absorbent tip of a microsampling device with a lysisbuffer and Proteinase K, and detecting the at least one mutation in thesample CFTR nucleic acid using high throughput massive parallelsequencing. In some embodiments, the lysis buffer comprises guanidinehydrochloride, Tris.Cl, EDTA, Tween 20, and Triton X-100.

Additionally or alternatively, in some embodiments, the plurality oftarget segments of the sample CFTR nucleic acid comprise at least onealteration compared to the corresponding region of a reference CFTRnucleotide sequence. A reference CFTR nucleotide sequence may be a CFTRgenomic or cDNA sequence, or a portion thereof, from a normal(non-cystic fibrosis afflicted and non-cystic fibrosis carrier)individual. In some cases, a reference CFTR sequence may comprise awild-type CFTR nucleic acid sequence. Various methods known in the art(e.g., read depth approach) can be employed to analyze sequencing datato determine if differences are present in the sample CFTR nucleic acidsequence compared to a reference CFTR nucleic acid sequence.

In some embodiments, the at least one mutation in the sample CFTRnucleic acid is selected from a base change, a gene deletion and a geneduplication. In some embodiments, the at least one mutation in thesample CFTR nucleic acid is associated with cystic fibrosis, and mayinclude more or more mutations disclosed in Table 2.

In some embodiments, the methods disclosed herein can be used to detectone or more rare CFTR mutations or private mutations in a CFTR samplenucleic acid obtained from an individual, thereby identifying anindividual who possesses one or more rare or private CFTR mutation(s).In some embodiments, the methods of the present technology are used toidentify rare familial mutations in an obligate cystic fibrosis carrierafter the carrier has tested negative in a routine screening test forcommon mutations. Such routine screening tests may include CF MutationScreen (Quest Diagnostics), CFTR Screen, Cystic Fibrosis Screen (QuestDiagnostics), and Cystic Fibrosis Carrier Screen (LabCorp). In someembodiments, the present methods can also be used to identify raremutations in a cystic fibrosis-affected (i.e. symptomatic) individualwho has not had two CFTR sequence mutations identified by at least oneroutine cystic fibrosis mutation screening test.

In one aspect, the present disclosure provides a method for detecting agenetic basis for being affected with cystic fibrosis, or for being acystic fibrosis carrier in an individual comprising: (a)generating anamplicon library by amplifying multiple target segments of a CFTRnucleic acid obtained from the individual, wherein the sample CFTRnucleic acid is extracted from a dried biological fluid sample elutedfrom an absorbent tip of a microsampling device; (b) sequencing theamplicons in the amplicon library using high throughput massive parallelsequencing, and (c) detecting a genetic basis for being affected withcystic fibrosis, or for being a cystic fibrosis carrier when thenucleotide sequence of one or more of the target segments of the CFTRnucleic acid comprises a mutation associated with cystic fibrosis.

In some embodiments, the methods disclosed herein are employed toconfirm cystic fibrosis carrier status in an individual such as, forexample, a parent, a sibling or other relatives of a cysticfibrosis-affected individual with one or more rare or private mutations.In some embodiments, the at least one mutation is associated with cysticfibrosis, and includes one or more mutations disclosed in Table 2. Bothgene sequence and gene dosage may be determined in a nucleic acid sampleusing the methods disclosed herein. Gene dosage (also referred to ascopy number variation or CNVs) can be determined by performing nextgeneration sequencing and using a read depth approach. CNVs are gainsand losses of genomic sequence >50 bp between two individuals of aspecies (Mills et al., Nature 470: 59-65 (2011)). A normal dosage inrelation to all other amplicons for a normal specimen will be one, ½ fora homozygous deletion and 1½ for a homozygous duplication.

In some embodiments, at least 2, 5, 10, 20, 25, or 28 and up to 25, 29,or 30, target segments of the CFTR gene may be sequenced with gains andlosses of genomic sequence (>50 bp) determined using a read depthapproach. In one embodiment, 29 target segments are sequenced,representing the CFTR coding region (including all exons/intronjunctions). In another embodiment, the CFTR coding region (including allexons/intron junctions) in addition to about 1 kb upstream and about 300kb downstream of the CFTR gene are assayed.

Additionally or alternatively, in some embodiments, each CFTR nucleicacid target segment may be amplified with an oligonucleotide primer orprimer pair specific to the target segment. In some embodiments a singleprimer or both primers of a primer pair comprise a specific adaptersequence (also referred to as a sequencing adapter) ligated to the 5′end of the target specific sequence portion of the primer. Thissequencing adapter is a short oligonucleotide of known sequence that canprovide a priming site for both amplification and sequencing of theadjoining, unknown nucleic acid. As such, adapters allow binding of afragment to a flow cell for next generation sequencing. Any adaptersequence may be included in a primer used in the present technology. Insome embodiments, the amplicons corresponding to the plurality of targetsegments of the sample CFTR nucleic acid are generated using primersthat contain an oligonucleotide sequencing adapter to produce adaptertagged amplicons. In other embodiments, the employed primers do notcontain adapter sequences and the amplicons produced are subsequently(i.e. after amplification) ligated to an oligonucleotide sequencingadapter on one or both ends of the amplicons.

In some embodiments, all forward amplicons (i.e., amplicons extendedfrom forward primers that hybridized with antisense strands of a targetsegment) contain the same adapter sequence. In some embodiments whendouble stranded sequencing is performed, all forward amplicons containthe same adapter sequence and all reverse amplicons (i.e., ampliconsextended from reverse primers that hybridized with sense strands of atarget segment) contain an adapter sequence that is different from theadapter sequence of the forward amplicons. In some embodiments, theadapter sequences further comprise an index sequence (also referred toas an index tag, a “barcode” or a multiplex identifier (MID)).

In some embodiments, the adapter sequences are P5 and/or P7 adaptersequences that are recommended for Illumina sequencers (MiSeq andHiSeq). See, e.g., Williams-Carrier et al., Plant J., 63(1):167-77(2010). In some embodiments, the adapter sequences are P1, A, or IonXpress™ barcode adapter sequences that are recommended for LifeTechnologies sequencers. Other adapter sequences are known in the art.Some manufacturers recommend specific adapter sequences for use with theparticular sequencing technology and machinery that they offer.

Additionally or alternatively, in some embodiments, the ampliconscorresponding to the plurality of target segments of the sample CFTRnucleic acid from more than one sample are sequenced. In someembodiments, all samples are sequenced simultaneously in parallel. Inany of the above embodiments, the amplicons corresponding to theplurality of target segments of the sample CFTR nucleic acid from atleast 1, 5, 10, 20, 30 or up to 35, 40, 45, 48 or 50 different samplesare amplified and sequenced using the methods described herein.

In some embodiments, amplicons derived from a single sample sourcefurther comprise an identical index sequence that indicates the sourcefrom which the amplicon is generated, the index sequence for each samplebeing different from the index sequences from all other samples. Assuch, the use of index sequences permits multiple samples to be pooledper sequencing run and the sample source subsequently ascertained basedon the index sequence. In some embodiments, the Access Array™ System(Fluidigm Corp., San Francisco, Calif.) or the Apollo 324 System(Wafergen Biosystems, Fremont, Calif.) is used to generate a barcoded(indexed) amplicon library by simultaneously amplifying the nucleicacids from the samples in one set up.

In some embodiments, indexed amplicons are generated using primers (forexample, forward primers and/or reverse primers) containing the indexsequence. Such indexed primers may be included during librarypreparation as a “barcoding” tool to identify specific amplicons asoriginating from a particular sample source. When adapter-ligated and/orindexed primers are employed, the adapter sequence and/or index sequencegets incorporated into the amplicon (along with the target-specificprimer sequence) during amplification. Therefore, the resultingamplicons are sequencing-competent and do not require the traditionallibrary preparation protocol. Moreover, the presence of the index tagpermits the differentiation of sequences from multiple sample sources.

In some embodiments, the amplicons may be amplified withnon-adapter-ligated and/or non-indexed primers and a sequencing adapterand/or an index sequence may be subsequently ligated to one or both endsof each of the resulting amplicons. In some embodiments, the ampliconlibrary is generated using a multiplexed PCR approach.

Indexed amplicons from more than one sample source are quantifiedindividually and then pooled prior to high throughput sequencing. Assuch, the use of index sequences permits multiple samples (i.e., samplesfrom more than one sample source) to be pooled per sequencing run andthe sample source subsequently ascertained based on the index sequence.“Multiplexing” is the pooling of multiple adapter-tagged and indexedlibraries into a single sequencing run. When indexed primer sets areused, this capability can be exploited for comparative studies. In someembodiments, amplicons from more than one sample source are pooled priorto high throughput sequencing. In some embodiments, amplicon librariesfrom up to 48 separate sources are pooled prior to sequencing.

In some embodiments, sequencing templates (amplicons) are prepared byemulsion-based clonal amplification of target segments using specializedfusion primers (containing an adapter sequence) and capture beads. Asingle adapter-bound fragment is attached to the surface of a bead, andan oil emulsion containing necessary amplification reagents is formedaround the bead/fragment component. Parallel amplification of millionsof beads with millions of single strand fragments produces asequencer-ready library.

Additionally or alternatively, in some embodiments the ampliconsconstituting the adapter-tagged (and, optionally, indexed) ampliconlibrary are produced by polymerase chain reaction (PCR). In someembodiments, the amplicon library is generated using a multiplexed PCRapproach, such as that disclosed in U.S. Pat. No. 8,092,996,incorporated by reference herein in its entirety.

Bridge PCR is yet another method for in vitro clonal amplification aftera library is generated, in preparation for sequencing. This process is ameans to clonally amplify a single target molecule, a member of alibrary, in a defined physical region such as a solid surface, forexample, a bead in suspension or a cluster on a glass slide. In thismethod, fragments are amplified using primers attached to the solidsurface forming “DNA colonies” or “DNA clusters.” This method is used insome of the genome analyzer sequencers manufactured by Illumina, Inc.(San Diego, Calif.).

Additionally or alternatively, in some embodiments, the plurality ofamplicons are enriched using a bait set comprising nucleic acidsequences that are complementary to at least one of the plurality ofamplicons. In some embodiments, the nucleic acid sequences of the baitset are RNA baits, DNA baits, or a combination thereof.

Following the production of an amplicon library, the amplicons aresequenced using high throughput, massive parallel sequencing (i.e. nextgeneration sequencing). Methods for performing high throughput,massively parallel sequencing are known in the art. In certainembodiments, the high throughput massive parallel sequencing isperformed using pyrosequencing, reversible dye-terminator sequencing,SOLiD sequencing, Ion semiconductor sequencing, Helioscope singlemolecule sequencing, sequencing by synthesis, sequencing by ligation, orSMRT™ sequencing.

Treatment of Cystic Fibrosis

Disclosed herein are methods for determining whether a patient willbenefit from one or more treatment for cystic fibrosis.

Examples of treatment for cystic fibrosis are well known in the art andinclude therapies that control the infectious microbiome in a patient'ssystem, such as treatment with antibiotics or anti-inflammatorymedications, chest physical therapies (CPTs), airway clearancetechniques (ACTs) and medications, nutrition therapies, organtransplantation (e.g., lung replacement surgery), etc.

Suitable antibiotics or combination of antibiotics may be used to treatinfections associated with cystic fibrosis. Classes of antibiotics thatare useful in the treatment of cystic fibrosis include Penicillins suchas penicillin and amoxicillin, Cephalosporins such as cephalexin(Keflex), Macrolides such as erythromycin (E-Mycin), clarithromycin(Biaxin), and azithromycin (Zithromax), Fluoroquinolones such asciprofolxacin (Cipro), levofloxacin (Levaquin), and ofloxacin (Floxin),Sulfonamides such as co-trimoxazole (Bactrim) and trimethoprim(Proloprim), Tetracyclines such as tetracycline (Sumycin, Panmycin) anddoxycycline (Vibramycin), Aminoglycosides such as gentamicin (Garamycin)and tobramycin (Tobrex), and Colistin.

Anti-inflammatory medications may be used to reduce inflammation andpain caused by cystic fibrosis associated infections. Classes ofanti-inflammatory medications include steroid-based anti-inflammatoryagents, such as Amcinonide, Betamethosone diproprionate, Clobetasol,Clocortolone, Dexamethasone, Diflorasone, Dutasteride, FlumethasonePivalate, Flunisolide, Fluocinolone Acetonide, Fluocinonide,Fluorometholone, Fluticasone propionate, Fluticasone propionate,Fluticasone propionate, Flurandrenolide and Hydroflumethiazide.Non-steroidal anti-inflammatory drugs (NSAIDs) include aceclofenac,acemetacin, aspirin, celecoxib, dexibuprofen, dexketoprofen, diclofenac,etodolac, etoricoxib, fenoprofen, flurbiprofen, ibuprofen, indometacin,ketoprofen, mefenamic acid, meloxicam, nabumetone, naproxen, sulindac,tenoxicam, and tiaprofenic acid.

Mucus thinning medications may be used to help keep a patient's lung andairway clear. Classes of mucus thinning medication include expectorants,antihistamines, and cough suppressants, such as Guaifenesin,Dextromethorphan, hypertonic salines, dornase alfa and mucolytics.

Chest physical therapies (CPTs) may involve chest clapping orpercussion. Pounding a patient's chest and back repeatedly may helploosening and dislodging the mucus from the lungs so that the patientmay cough up the mucus. In some cystic fibrosis therapeutic regimens,CPT is performed on the patient three to four times a day. In somecystic fibrosis therapeutic regimens, the patients may sit or lie ontheir stomach while CPT is performed to facilitate drainage of the mucusfrom the lungs. Certain devices may be used in CPT to reduce thepatient's discomfort during the process. Exemplary devices include, butare not limited to, an electric chest clapper, an inflatable therapyvest that uses high-frequency air waves to force the mucus out of thelungs, a flutter device that a patient uses to breath out through,causing vibrations that dislodge the mucus, and a positive expiratorypressure mask that creates back pressure to help hold airways open,again facilitating dislodging of mucus from the airway walls.

Airway clearance techniques (ACTs) may help to loosen thick, stickymucus so it can be cleared from patients' lungs by coughing or huffing.Clearing the airways may help decrease lung infections and improve lungfunction. Various ACTs are known and clinically performed. For example,coughing is a basic airway clearance technique. Coughing may be aninvoluntary reflex or can be controlled as a healthy, natural way forthe lungs to eliminate mucus. Additionally, several breathing techniquesmay also help clear the patient's airway. Examples of these techniquesinclude forced expiration technique (FET) which involves forcing out acouple of breaths of huffs followed by relaxed breathing; and activecycle breathing (ACB) that involves deep breathing exercises that canloosen the mucus and help open airways. In some cystic fibrosistherapeutic regimens, ACTs are used with other treatments, includinginhaling medications that help relax airway wall muscles, and thin anddislodge mucus. Such medications may include but are not limited tobronchodilators, antibiotics, and mucus thinners. In some embodiments,medications are taken through a nebulizer during ACTs.

Nutritional therapy may be used alone or in combination with othertherapies for treating cystic fibrosis. For example, a patient mayreceive pancreatic enzymes that aids in the digestion of fats andprotein, and absorption of vitamins. Vitamin A, D, E, and K supplementsmay be administered to the patient to provide an additional source offat-soluble vitamins. Feeding tubes such as a gastrostomy tube (G-tube),may be used to feed nutritional solutions directly to the patient'sstomach. Medications or supplements that may reduce stomach acid may beadministered concurrently with oral pancreatic enzymes. Other mucusthinners may be administered individually or concurrently to treatintestinal blockage as part of a nutritional therapy by correctingdigestive problems.

In one aspect, the present disclosure provides a method for selecting apatient exhibiting cystic fibrosis symptoms, or a patient at risk forcystic fibrosis for treatment with an anti-cystic fibrosis therapeuticagent comprising (a) eluting a dried biological fluid sample of thepatient from an absorbent tip of a microsampling device, wherein thedried biological fluid sample comprises a sample CFTR nucleic acid; (b)generating a library comprising amplicons corresponding to a pluralityof target segments of the sample CFTR nucleic acid; (c) detecting atleast one mutation in at least one of the amplicons in the library usinghigh throughput massive parallel sequencing; and (d) selecting thepatient for treatment with an anti-cystic fibrosis therapeutic agent.The dried biological fluid sample may be dried plasma, dried serum, ordried whole blood. In some embodiments, the microsampling device is avolumetric absorbent microsampling device. In certain embodiments, thedried biological fluid sample on the absorbent tip of the microsamplingdevice is collected from a patient via fingerstick. In certainembodiments, the microsampling device is a MITRA® tip. In someembodiments, the patient harbors one or more mutations in the CFTR geneand may include one or more mutations listed in Table 2.

Patients at risk for cystic fibrosis include subjects having: (a) agenetic basis for cystic fibrosis; (b) at least one parent or at leastone grandparent having a genetic basis for cystic fibrosis, such asbeing a cystic fibrosis carrier; (c) familial incidences of cysticfibrosis in multiple generations; or (d) one or more symptoms possiblyrelating to or caused by cystic fibrosis.

Cystic fibrosis related symptoms include, but are not limited to,perturbations in the body's secretion of mucus and sweat, as well asassociated complications and symptoms in the respiratory system,digestive system, and reproductive system. For example, a cysticfibrosis patient may routinely exhibit large amounts of thick, sticky,sometimes bloody mucus accumulating in the lung and airways. Thisbuildup of mucus may result in coughing, wheezing or shortness ofbreath, and can also make it easier for bacteria to cause infections inthe respiratory system. An infection caused by unusual pathogens that donot respond to standard antibiotics, such as lung infections caused bymucoid Pseudomonas, may be a sign of cystic fibrosis. Additional cysticfibrosis related symptoms may include sinus infections, bronchitis orpneumonia, growths (i.e., polyps) in the nose.

A cystic fibrosis patient may exhibit mucus obstructed tubes in thepancreas. These blockages prevent the delivery of digestive enzymes tothe digestive tract, which results in impaired digestion and absorption.Accordingly, cystic fibrosis related symptoms may also include weightloss or failure to gain weight, ongoing diarrhea or bulky,foul-smelling, greasy stools, intestinal blockages, excess gas,constipation, stomach pain and discomfort. In the long term, thesedigestive system complications could result in malnutrition,pancreatitis, rectal prolapse, liver diseases, diabetes, and gallstones.

Cystic fibrosis related signs and symptoms may also include infertility,low bone density and related bone-thinning disorders such asosteoporosis, very salty sweat, dehydration, bodily fluid imbalance andensuing increased heart rate, fatigue, weakness, low blood pressure,heat stroke, and widening and rounding of fingertips and toes known asclubbing.

In some embodiments, the treatment of cystic fibrosis comprises one ormore of infection control therapy, chest physical therapy, airwayclearance therapy, nutrition therapy, and organ transplantation.

In any of the above embodiments, the anti-cystic fibrosis therapeuticagent is one or more agents selected from the group consisting ofpenicillin, amoxicillin, cephalosporins, macrolides, fluoroquinolones,sulfonamides, Tetracyclines, aminoglycosides, colistin, Amcinonide,Betamethosone diproprionate, Clobetasol, Clocortolone, Dexamethasone,Diflorasone, Dutasteride, Flumethasone Pivalate, Flunisolide,Fluocinolone Acetonide, Fluocinonide, Fluorometholone, Fluticasonepropionate, Fluticasone propionate, Fluticasone propionate,Flurandrenolide, Hydroflumethiazide, aceclofenac, acemetacin, aspirin,celecoxib, dexibuprofen, dexketoprofen, diclofenac, etodolac,etoricoxib, fenoprofen, flurbiprofen, ibuprofen, indometacin,ketoprofen, mefenamic acid, meloxicam, nabumetone, naproxen, sulindac,tenoxicam, tiaprofenic acid, expectorants, antihistamines, coughsuppressants, Dextromethorphan, hypertonic salines, dornase alfa,mucolytics, pancreatic enzymes, vitamin A, vitamin D, vitamin E, vitaminK, and supplements reduce stomach acid.

Kits

The present disclosure provides kits for detecting one or more mutationsin a sample CFTR nucleic acid in a dried biological fluid sample. Insome embodiments, the kits comprise a skin puncture tool, a volumetricabsorptive microsampling device, a lysis buffer, and proteinase K,wherein the one or more mutations comprises one or more mutations listedin Table 2. The lysis buffer may comprise guanidine hydrochloride,Tris.Cl, EDTA, Tween 20, and Triton X-100. In other embodiments, thelysis buffer comprises 2.5-10% sodium dodecyl sulphate.

In some embodiments, the kits further comprise one or more componentsfor denaturing nucleoprotein complexes in cells present in the driedbiological fluid sample. Additionally or alternatively, in someembodiments, the kits further comprise one or more components forremoving protein contaminants, inactivating nuclease activity, and/orremoving biological and/or chemical contaminants present in the driedbiological fluid sample.

In some embodiments, the kits further comprise one or more primer pairsthat hybridize to one or more target segments of the sample CFTR nucleicacid. Additionally or alternatively, in some embodiments, the kitsfurther comprise one or more bait sequences that hybridize to one ormore target segments of the sample CFTR nucleic acid. In someembodiments, the target segments of the sample CFTR nucleic acidcorrespond to a coding or non-coding region of the CFTR gene, such as anexon or intron region. In some embodiments, the target segments of thesample CFTR nucleic acid correspond to a regulatory sequence of the CFTRregion, such as a CFTR promoter region or a downstream regulatoryregion. In some embodiments, the target segments of the sample CFTRnucleic acid may include one or more CFTR mutations listed in Table 2.

Particularly, in some embodiments, kits of the present technologycomprise one or more primer pairs or bait sequences that selectivelyhybridize to, and are useful in amplifying or capturing one or moretarget segments of the sample CFTR nucleic acid. Particularly, in someembodiments, the target segments of the sample CFTR nucleic acidcorrespond to a coding or non-coding region of the CFTR gene, such as anexon or intron region. In some embodiments, the target segments of thesample CFTR nucleic acid correspond to a regulatory sequence of the CFTRregion, such as a CFTR promoter region or a downstream regulatoryregion. In some embodiments, the target segments of the sample CFTRnucleic acid may include one or more CFTR mutations listed in Table 2.

In some embodiments, the kits of the present technology comprise asingle primer pair or bait sequence that hybridizes to one or moretarget segments of the sample CFTR nucleic acid. Examples of usefulprimer pairs may be found in PCT/US2014/027870, which is hereinincorporated by reference in its entirety. In other embodiments, thekits of the present technology comprise multiple primer pairs or baitsequences that hybridize to multiple target segments of the sample CFTRnucleic acid. In certain embodiments, the kits of the present technologycomprise multiple primer pairs or bait sequences comprising more thanone primer pair or more than one bait sequence that hybridizes to one ormore target segments of the sample CFTR nucleic acid. In someembodiments, the target segments of the sample CFTR nucleic acidcorrespond to a coding or non-coding region of the CFTR gene, such as anexon or intron region. In some embodiments, the target segments of thesample CFTR nucleic acid correspond to a regulatory sequence of the CFTRregion, such as a CFTR promoter region or a downstream regulatoryregion. Thus, it is contemplated herein that the kits of the presenttechnology can comprise primer pairs or bait sequences that recognizeand specifically hybridize to one or more target segments of a sampleCFTR nucleic acid.

In any of the above embodiments of the kits of the present technology,the volumetric absorptive microsampling device is a MITRA® tip.

In some embodiments, the kits may comprise a plurality of volumetricabsorptive microsampling devices, each having a hollow holder at theproximal end and an absorbent tip at the distal end. The absorbent tipcomprises a hydrophilic, polymeric material configured to absorb 30microliters or less of blood within about 10 seconds or less. The kitalso includes a container having a plurality of compartments. Eachcompartment is configured to releasably engage a volumetric absorptivemicrosampling device. The container is configured to prevent theabsorbent tips of the microsampling devices from abutting thecompartment within which the microsampling device is placed.

Additionally or alternatively, in certain embodiments, the kits mayinclude a plurality of access ports with each port associated with anindividual compartment. Each port is located to allow printing onto theholder of a volumetric absorptive microsampling device present withinthe compartment with which the port is associated. In certainembodiments, the holder of a volumetric absorptive microsampling devicehas a plurality of ribs extending along a length of the holder with theribs configured to keep the absorbent tip from contacting walls of thecontainer. The container preferably has two parts configured to formtubular shaped compartments. The container may have a first part with aplurality of elongated mounting protrusions each extending along aportion of a different compartment. The hollow end of the holder of thevolumetric absorptive microsampling device fits onto the mountingprotrusion to releasably fasten the holder onto the mounting protrusion.

In some embodiments, the kits further comprise buffers, enzymes havingpolymerase activity, enzymes having polymerase activity and lacking5′→3′ exonuclease activity or both 5′→3′ and 3′→5′ exonuclease activity,enzyme cofactors such as magnesium or manganese, salts, chain extensionnucleotides such as deoxynucleoside triphosphates (dNTPs), modifieddNTPs, nuclease-resistant dNTPs or labeled dNTPs, necessary to carry outan assay or reaction, such as amplification and/or detection of one ormore hereditary cystic fibrosis mutations described herein, in a driedbiological fluid sample.

In one embodiment, the kits of the present technology further comprise apositive control nucleic acid sequence and a negative control nucleicacid sequence to ensure the integrity of the assay during experimentalruns. The kit may also comprise instructions for use, software forautomated analysis, containers, packages such as packaging intended forcommercial sale and the like.

The kit may further comprise one or more of: wash buffers and/orreagents, hybridization buffers and/or reagents, labeling buffers and/orreagents, and detection means. The buffers and/or reagents are usuallyoptimized for the particular amplification/detection technique for whichthe kit is intended. Protocols for using these buffers and reagents forperforming different steps of the procedure may also be included in thekit.

The kits of the present technology may include components that are usedto prepare nucleic acids from a dried biological fluid sample for thesubsequent amplification and/or detection of alterations in a sampleCFTR nucleic acid (e.g., CFTR mutations disclosed in Table 2). Suchsample preparation components can be used to produce nucleic acidextracts from dried biological fluid samples, such as dried serum, driedplasma, or dried whole blood. The test samples used in theabove-described methods will vary based on factors such as the assayformat, nature of the detection method, and the specific cells orextracts used as the test sample to be assayed. Methods of extractingnucleic acids from samples are well known in the art and can be readilyadapted to obtain a sample that is compatible with the system utilized.Automated sample preparation systems for extracting nucleic acids from atest sample are commercially available, e.g., Roche Molecular Systems'COBAS AmpliPrep System, Qiagen's BioRobot 9600, Qiagen's BioRobot EZ1,QIAsymphony, and Applied Biosystems' PRISM™ 6700 sample preparationsystem.

EXAMPLES Example 1 Extraction of Genomic DNA from Dried Blood SamplesCollected Using MITRA® Tips

This Example demonstrates that the methods of the present technology areuseful for extracting high yields of genomic DNA from a dried biologicalfluid sample (e.g., dried blood) collected using a volumetric absorptivemicrosampling device.

A total of four human subjects were enrolled in the study. Three MITRA®tips of blood were collected from each of 4 blood donors in order tosimultaneously test three extraction methods. A fixed volume of 10 μL ofblood was collected on each MITRA® Tip collection device viafingerstick. After drying the blood samples, the absorbent tips of theMITRA® Tip collection devices were then placed in 180 μL Buffer G2 (alysis buffer containing 800 mM guanidine hydrochloride; 30 mM Tris.Cl,pH 8.0; 30 mM EDTA, pH 8.0; 5% Tween 20; 0.5% Triton X-100) and werevortexed for 15 seconds. The remaining sample processing steps for eachof the three extraction methods are summarized below:

Step Method 1 Method 2 Method 3 1 Incubate MITRA ® Tip in — IncubateMITRA ® Tip in Buffer G2 at 90° C. for 15 min Buffer G2 at 90° C. for 15min 2 Vortex for 15 sec — Vortex for 15 sec 3 Add 10 μL Proteinase K 4Vortex for 15 sec 5 Incubate with Proteinase Incubate with ProteinaseIncubate with Proteinase K at 56° C. for 1 hour K at 56° C. for 1 hour Kat 56° C. Overnight 6 Vortex 15 sec 7 Aliquot cell lysate to new tube 8Perform remaining genomic DNA extraction on EZ1 ® Biorobot using TissueDNA protocol

Extracted genomic DNA was then quantified using Qubit dsDNA HS AssayKit, which uses a dsDNA intercalating dye that only fluoresces in thepresence of dsDNA. Therefore, quantitation of dsDNA using the Qubit°dsDNA HS Assay Kit is not affected by RNA, proteins, salts, or othercontaminants that may affect other quantitation methods. Table 1demonstrates that the DNA yield obtained from each MITRA® tip variedaccording to the extraction method. It was determined that extractionmethod 3 (Incubation of MITRA® Tip with Buffer G2 at 90° C. for 15 min,and with Proteinase K at 56° C. overnight) yielded the highest quantityof DNA.

TABLE 1 Range of DNA yield obtained per MITRA ® Tip Extraction MethodTotal DNA yield (ng) 1 111-248 2 163-210 3 222-390

These results demonstrate that the methods of the present technology areuseful for extracting high yields of genomic DNA from a dried biologicalfluid sample (e.g., dried blood) collected using a volumetric absorptivemicrosampling device.

Example 2 Comparison of DNA Yield Using One-Tip and Dual-Tip Extractions

DNA extractions were performed on lysates eluted from a single MITRA®tip (one-tip extraction) containing dried blood derived from anindividual patient. For dual-tip extractions, lysates eluted from twoindividual MITRA® tips containing dried blood derived from the samepatient were combined together. MITRA® tips were incubated with BufferG2 at 90° C. for 15 min, and with Proteinase K at 56° C. overnight.

Subsequent nucleic acid extraction was performed using the DNAInvestigator kit on the QIAsymphony® automated extraction platformaccording to the manufacturer's instructions. DNA yields from theone-tip and dual-tip extractions were compared (See FIG. 1).

These results demonstrate that dual-tip extraction on average resultedin a 2-fold increase in DNA yield (FIG. 1). These results demonstratethat the methods of the present technology are useful for extractinghigh yields of genomic DNA from a dried biological fluid sample (e.g.,dried blood) collected using a volumetric absorptive microsamplingdevice.

Example 3 CFvantage® Cystic Fibrosis Expanded Screen using Dried BloodSamples Extracted from MITRA® Tips

Genomic DNA was extracted from dried blood specimens obtained from 7donors. Each extraction was performed using a dried blood specimencollected using a single MITRA® tip, i.e., one-tip extraction asdescribed in Example 2. Extracted DNA was then tested on the CFvantage®Cystic Fibrosis Expanded Panel, which assesses 38 ampliconscorresponding to the CFTR gene. The CFvantage® Cystic Fibrosis ExpandedPanel covers 162 CFTR mutations that are associated with cystic fibrosis(Table 2). Next generation sequencing was performed on the MiSeqSequencer. Quality metrics assessing successful sequencing of a coveredregion include a minimum of 30 reads per covered hotspot.

Results: As shown in FIG. 2, all 7 samples passed the QC criteria for100% of the covered hotspot regions.

These results demonstrated that the methods of the present technologyare capable of detecting at least one mutation in a sample CFTR nucleicacid in a small-volume dried biological fluid sample that is collectedwith a volumetric absorptive microsampling device (e.g., MITRA® Tip).

TABLE 2 CF Mutations Detected in the CFvantage ® Cystic FibrosisExpanded Screen Conventional HGVS cDNA Name Nomenclature 296 + 2T > Ac.164 + 2T > A 394delTT c.262_263delTT 405 + 1G > A c.273 + 1G > A 406 −1G > A c.274 − 1G > A 444delA c.313delA 457TAT > G c.325_327delTATinsG574delA c.442delA 621 + 1G > T c.489 + 1G > T 663delT c.531delT 711 +1G > T c.579 + 1G > T 711 + 3A > G c.579 + 3A > G 711 + 5G > A c.579 +5G > A 712 − 1G > T c.580 − 1G > T 852del22 c.720_741del22 935delAc.803delA 936delTA c.805_806delAT 1078delT c.948delT 1154insTCc.1022_1023insTC 1161delC c.1029delC 1213delT c.1081delT 1248 + 1G > Ac.1116 + 1G > A 1259insA c.1127_1128insA 1288insTA c.1153_1154insAT1341 + 1G > A c.1209 + 1G > A 1461ins4 c.1329_1330insAGAT 1525 − 1G > Ac.1393 − 1G > A 1548delG c.1418delG 1609delCA c.1477_1478delCA 1677delTAc.1545_1546delTA 1717 − 1G > A c.1585 − 1G > A 1717 − 8G > A c.1585 −8G > A 1811 + 1.6kbA > G c.1679 + 1.6kbA > G 1812 − 1G > A c.1680 − 1G >A 1898 + 1G > A c.1766 + 1G > A 1898 + 1G > T c.1766 + 1G > T 1898 +3A > G c.1766 + 3A > G 1898 + 5G > T c.1766 + 5G > T 2043delG c.1911delG2055del9 > A c.1923_1931del9insA 2105del13ins5 c.1973_1985del13insAGAAA2108delA c.1976delA 2143delT c.2012delT 2183AA > G c.2051_2052delAAinsG2184delA c.2052delA 2184insA c.2052_2053insA 2307insA c.2175_2176insA2347delG c.2215delG 2585delT c.2453delT 2622 + 1G > A c.2490 + 1G > A2711delT c.2583delT 2789 + 5G > A c.2657 + 5G > A 2869insGc.2737_2738insG 3007delG c.2875delG 3120 + 1G > A c.2988 + 1G > A3120G > A c.2988G > A 3121 − 1G > A c.2989 − 1G > A 3171delC c.3039delC3199del6 c.3067_3072delATAGTG 3272 − 26A > G c.3140 − 26A > G 3659delCc.3528delC 3667del4 c.3535_3538delACCA 3791delC c.3659delC 3821delTc.3691delT 3849 + 10kbC > T c.3717 + 12191C > T 3876delA c.3744delA3905insT c.3773_3774insT 4005 + 1G > A c.3873 + 1G > A 4016insTc.3884_3885insT 4209TGTT > AA c.4077_4080delTGTTinsAA 4382delAc.4251delA A455E c.1364C > A A559T c.1675G > A C524X c.1572C > ACFTRdele2,3 c.54-5940_273 + 10250del21kb CFTRdele22,23 c.3964-78_4242 +577del D110H c.328G > C D579G c.1736A > G E60X c.178G > T E92K c.274G >A E92X c.274G > T E585X c.1753G > T E822X c.2464G > T E831X c.2491G > TE1104X c.3310G > T F311del c.933_935delCTT F508del c.1521_1523delCTTG85E^(a) c.254G > A G91R c.271G > A G178R c.532G > A G330X c.988G > TG480C c.1438G > T G542X c.1624G > T G551D c.1652G > A G970R c.2908G > CG1244E c.3731G > A H199Y c.595C > T I336K c.1007T > A I507delc.1519_1521delATC I1234V c.3700A > G K710X c.2128A > T L206W c.617T > GL467P c.1400T > C L732X c.2195T > G L927P c.2780T > C L1065P c.3194T > CL1077P c.3230T > C L1093P c.3278T > C M1V c.1A > G M1101K c.3302T > AN1303K c.3909C > G P67L c.200C > T P205S c.613C > T P574H c.1721C > AQ39X c.115C > T Q98X c.292C > T Q220X c.658C > T Q493X c.1477C > T Q525Xc.1573C > T Q552X c.1654C > T Q890X c.2668C > T Q1238X c.3712C > TQ1313X c.3937C > T R75X c.223C > T R117C c.349C > T R117H c.350G > AR334W c.1000C > T R347H c.1040G > A R347P c.1040G > C R352Q c.1055G > AR553X c.1657C > T R560K c.1679G > A R560T c.1679G > C R709X c.2125C > TR764X c.2290C > T R851X c.2551C > T R1066C c.3196C > T R1066H c.3197G >A R1128X c.3382A > T R1158X c.3472C > T R1162X c.3484C > T R1283Mc.3848G > T S341P c.1021T > C S466X c.1397C > A or c.1397C > G S489Xc.1466C > A S492F c.1475C > T S549N c.1646G > A S549R c.1645A > C orc.1647T > G S945L c.2834C > T S1196X c.3587C > G S1251N c.3752G > AS1255X c.3764C > A T338I c.1013C > T V520F c.1558G > T W401X c.1202G > Aor c.1203G > A W846X c.2537G > A W1089X c.3266G > A W1145X c.3435G > AW1204X c.3611G > A or c.3612G > A W1282X c.3846G > A Y122X c.366T > AY1092X c.3276C > A or c.3276C > G S364P c. 1090T > C

Equivalents

The present technology is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods andapparatuses within the scope of the present technology, in addition tothose enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the present technology. It is to beunderstood that this present technology is not limited to particularmethods, reagents, compounds compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

The terms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the disclosure claimed.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

What is claimed is:
 1. A method for detecting at least one mutation in asample CFTR nucleic acid comprising (a) extracting the sample CFTRnucleic acid from a dried biological fluid sample eluted from anabsorbent tip of a microsampling device; (b) generating a librarycomprising amplicons corresponding to a plurality of target segments ofthe sample CFTR nucleic acid; and (c) detecting at least one mutation inat least one of the amplicons in the library using high throughputmassive parallel sequencing.
 2. The method of claim 1, wherein the driedbiological fluid sample is dried plasma, dried serum, or dried wholeblood.
 3. The method of claim 1 or 2, wherein the dried biological fluidsample on the absorbent tip of the microsampling device is collectedfrom a patient via fingerstick.
 4. The method of any one of claims 1 to3, wherein elution of the dried biological fluid sample is performed bycontacting the absorbent tip of the microsampling device with a lysisbuffer and Proteinase K.
 5. The method of claim 4, wherein the lysisbuffer comprises guanidine hydrochloride, Tris.Cl, EDTA, Tween 20, andTriton X-100.
 6. The method of claim 4 or 5, wherein elution of thedried biological fluid sample is performed by contacting the absorbenttip of the microsampling device with the lysis buffer for up to 15minutes at 90° C.
 7. The method of any one of claims 4-6, whereinelution of the dried biological fluid sample is performed by contactingthe absorbent tip of the microsampling device with Proteinase K for upto 1 hour at 56° C.
 8. The method of any one of claims 4-7, whereinelution of the dried biological fluid sample is performed by contactingthe absorbent tip of the microsampling device with Proteinase K for upto 16-18 hours at 56° C.
 9. The method of any one of claims 1 to 8,wherein the microsampling device is a volumetric absorbent microsamplingdevice.
 10. The method of any one of claims 1 to 9, wherein themicrosampling device is a MITRA® tip.
 11. The method of any one ofclaims 1 to 10, wherein the sample volume of the microsampling device isno more than 10-20 μL.
 12. The method of any one of claims 1-11, whereinno more than 400 ng of genomic DNA is eluted from the absorbent tip ofthe microsampling device.
 13. The method of any one of claims 1-12,wherein about 100 ng to about 400 ng of genomic DNA is eluted from theabsorbent tip of the microsampling device.
 14. The method of any one ofclaims 1-13, wherein the at least one mutation is selected from among abase change, a gene deletion and a gene duplication.
 15. The method ofany one of claims 1-14, wherein the at least one mutation is selectedfrom a mutation listed in Table
 2. 16. The method of any one of claims1-15, wherein the at least one mutation is associated with cysticfibrosis.
 17. The method of any one of claims 1-16, wherein the driedbiological fluid sample is obtained from an individual exhibiting cysticfibrosis symptoms, or having a family history of cystic fibrosis or aCFTR mutation.
 18. The method of any one of claims 1-17, wherein thedried biological fluid sample is obtained from a male partner of anobstetrics and gynecology patient having cystic fibrosis or at least oneCFTR mutation.
 19. The method of any one of claims 1-18, wherein theplurality of target segments, together, span all coding and non-codingregions of the CFTR gene.
 20. The method of claim 19, wherein theplurality of target segments further span about 1000 nucleotides of apromoter region immediately upstream of the first exon of the CFTR gene.21. The method of claim 20, wherein the plurality of target segmentsfurther span about 200 to 350 nucleotides immediately downstream of theCFTR gene.
 22. The method of any one of claims 1-21, wherein the highthroughput massive parallel sequencing is performed usingpyrosequencing, reversible dye-terminator sequencing, SOLiD sequencing,Ion semiconductor sequencing, Helioscope single molecule sequencing,sequencing by synthesis, sequencing by ligation, or SMRT™ sequencing.23. The method of any one of claims 1 to 22, wherein the high throughputmassive parallel sequencing involves a read depth approach.
 24. A methodfor detecting at least one mutation in a sample CFTR nucleic acidcomprising generating a library comprising amplicons corresponding to aplurality of target segments of the sample CFTR nucleic acid, whereinthe sample CFTR nucleic acid is extracted from a dried biological fluidsample eluted from an absorbent tip of a microsampling device with alysis buffer and Proteinase K.
 25. The method of claim 24, wherein theat least one mutation in the sample CFTR nucleic acid is detected usinghigh throughput massive parallel sequencing.
 26. The method of any oneof claims 1-25, wherein the plurality of target segments of the sampleCFTR nucleic acid comprise at least one alteration compared to thecorresponding region of a reference CFTR nucleotide sequence.
 27. Themethod of claim 26, wherein the reference CFTR nucleotide sequencecomprises a wild-type CFTR nucleic acid sequence.
 28. A method forselecting a patient exhibiting cystic fibrosis symptoms, or a patient atrisk for cystic fibrosis for treatment with an anti-cystic fibrosistherapeutic agent comprising (a) eluting a dried biological fluid sampleof the patient from an absorbent tip of a microsampling device, whereinthe dried biological fluid sample comprises a sample CFTR nucleic acid;(b) generating a library comprising amplicons corresponding to aplurality of target segments of the sample CFTR nucleic acid; (c)detecting at least one mutation in at least one of the amplicons in thelibrary using high throughput massive parallel sequencing; and (d)selecting the patient for treatment with an anti-cystic fibrosistherapeutic agent.
 29. The method of claim 28, wherein the anti-cysticfibrosis therapeutic agent is one or more agents selected from the groupconsisting of penicillin, amoxicillin, cephalosporins, macrolides,fluoroquinolones, sulfonamides, Tetracyclines, aminoglycosides,colistin, Amcinonide, Betamethosone diproprionate, Clobetasol,Clocortolone, Dexamethasone, Diflorasone, Dutasteride, FlumethasonePivalate, Flunisolide, Fluocinolone Acetonide, Fluocinonide,Fluorometholone, Fluticasone propionate, Fluticasone propionate,Fluticasone propionate, Flurandrenolide, Hydroflumethiazide,aceclofenac, acemetacin, aspirin, celecoxib, dexibuprofen,dexketoprofen, diclofenac, etodolac, etoricoxib, fenoprofen,flurbiprofen, ibuprofen, indometacin, ketoprofen, mefenamic acid,meloxicam, nabumetone, naproxen, sulindac, tenoxicam, tiaprofenic acid,expectorants, antihistamines, cough suppressants, Dextromethorphan,hypertonic salines, dornase alfa, mucolytics, pancreatic enzymes,vitamin A, vitamin D, vitamin E, vitamin K, and supplements reducestomach acid.
 30. A kit for detecting at least one mutation in a sampleCFTR nucleic acid in a dried biological fluid sample comprising a skinpuncture tool, a volumetric absorptive microsampling device, a lysisbuffer, and proteinase K, wherein the at least one mutation comprisesone or more of the CFTR mutations listed in Table
 2. 31. The kit ofclaim 30, further comprising one or more primer pairs that hybridize toone or more target segments of the sample CFTR nucleic acid.
 32. The kitof claim 30 or 31, further comprising one or more bait sequences thathybridize to one or more target segments of the sample CFTR nucleicacid.
 33. The kit of any one of claims 30-32, wherein the volumetricabsorptive microsampling device is a MITRA® tip.
 34. The kit of any oneof claims 30-33, wherein the lysis buffer comprises guanidinehydrochloride, Tris.Cl, EDTA, Tween 20, and Triton X-100.